The application of metagenomic second-generation sequencing (mNGS) is shifting from research to clinical laboratories due to rapid technological advances and significant cost reductions. Although many studies and case reports have confirmed that the success of mNGS in improving the prevention, diagnosis, treatment and tracking of infectious diseases, there are still some obstacles that must be overcome. The results of mNGS show all the possible pathogens in the sample, however, in the face of thousands of microbes that can infect humans, it remains challenging to accurately identify the key pathogens. So far, there is no unified interpretation standard for mNGS in clinical practice. This article reviews the interpretation of mNGS results for pathogen infection in different systems, the clinical interpretation and application regulations of mNGS results, and the challenges of mNGS interpretation in pathogen diagnosis.
Humans ; Communicable Diseases ; Metagenomics ; Sensitivity and Specificity

OBJECTIVE: To improve the understanding of the virome and bacterial microbiome in the wildlife rescue station of Poyang Lake, China. METHODS: Ten smear samples were collected in March 2019. Metagenomic sequencing was performed to delineate bacterial and viral diversity. Taxonomic analysis was performed using the Kraken2 and Bracken methods. A maximum-likelihood tree was constructed based on the RNA-dependent RNA polymerase (RdRp) region of picornavirus. RESULTS: We identified 363 bacterial and 6 viral families. A significant difference in microbial and viral abundance was found between samples S01-S09 and S10. In S01-S09, members of Flavobacteriia and Gammaproteobacteria were the most prevalent, while in S10, the most prevalent bacteria class was Actinomycetia. Among S01-S09, members of Myoviridae and Herelleviridae were the most prevalent, while the dominant virus family of S10 was Picornaviridae. The full genome of the pigeon mesivirus-like virus (NC-BM-233) was recovered from S10 and contained an open reading frame of 8,124 nt. It showed the best hit to the pigeon mesivirus 2 polyprotein, with 84.10% amino acid identity. Phylogenetic analysis showed that RdRp clustered into Megrivirus B. CONCLUSION This study provides an initial assessment of the bacteria and viruses in the cage-smeared samples, broadens our knowledge of viral and bacterial diversity, and is a way to discover potential pathogens in wild birds.
Animals ; Animals, Wild/genetics* ; Lakes ; Phylogeny ; Picornaviridae/genetics* ; Viruses/genetics* ; China ; Metagenomics ; Genome, Viral

In recent years, many studies have found that vaginal microbiota is closely related to female reproductive tract diseases. However, traditional microbial culture technology has the defects of long culture cycle and most microorganisms cannot be cultured. The development of metagenomics technique has broken the limitations of culture technology, and has been gradually applied to the study of vaginal microorganisms with the characteristics of high throughput, short time, identification of microbial population structure and gene function. It also provides technical support for elucidating the relationship between vaginal microbiota and female reproductive tract diseases. This article mainly introduces the metagenomics techniques and their applications in prevention, screening and diagnosis of common female reproductive tract diseases, and discusses their promising development and limitations to be overcome.
Female ; Humans ; Microbiota/genetics* ; Vagina ; Metagenomics/methods*

Infectious diseases are commonly seen in clinical practice, and pathogen diagnosis is the key link in diagnosis and treatment; however, conventional pathogen detection methods cannot meet clinical needs due to time-consuming operation and low positive rate. As a new pathogen detection method, metagenomic next-generation sequencing (mNGS) has a wide detection range and can detect bacteria, viruses, fungi, parasites, rare pathogens, and even unknown pathogens. The technique of mNGS is unbiased and can rapidly, efficiently, and accurately obtain all nucleic acid information in test samples, analyze pathogens, and guide clinical diagnosis and treatment, thereby playing an important role in complicated infectious diseases. This article reviews the diagnostic advantages and clinical value of mNGS in bacterial, fungal, viral, and parasitic infections.
Bacteria ; Communicable Diseases/diagnosis* ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Metagenomics/methods* ; Sensitivity and Specificity

OBJECTIVES: To study the application value of metagenomic next-generation sequencing (mNGS) in children with severe infectious diseases. METHODS: An analysis was performed on the clinical data and laboratory test results of 29 children with severe infection who were admitted to the Second Affiliated Hospital of Wenzhou Medical University from June 2018 to December 2020. Conventional pathogen culture was performed for the 29 specimens (27 peripheral blood specimens and 2 pleural effusion specimens) from the 29 children, and mNGS pathogen detection was performed at the same time. RESULTS: Among the 29 children, 2 tested positive by conventional pathogen culture with 2 strains of pathogen, and the detection rate was 7% (2/29); however, 20 children tested positive by mNGS with 38 strains of pathogen, and the detection rate was 69% (20/29). The pathogen detection rate of mNGS was significantly higher than that of conventional pathogen culture (P<0.05), and mNGS could detect the viruses, fungi, and other special pathogens that conventional pathogen culture failed to detect, such as Orientia tsutsugamushi. The univariate analysis showed that gender, routine blood test results, C-reactive protein, procalcitonin, D-dimer, radiological findings, and whether antibiotics were used before admission did not affect the results of mNGS (P>0.05). CONCLUSIONS Compared with conventional pathogen culture, mNGS is more sensitive for pathogen detection, with fewer interference factors. Therefore, it is a better pathogenic diagnosis method for severe infectious diseases in children.
Anti-Bacterial Agents ; Child ; Communicable Diseases ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Metagenomics/methods* ; Sensitivity and Specificity

Fecal microbiota transplantation (FMT) refers to using the intestinal microorganisms present in the feces or processed feces from healthy people for treating various types of diseases, such as digestive and metabolic diseases. The rapid development of metagenomic and culturomic technologies in gut microbiome analysis provides powerful tools for the FMT research and its clinical applications. Metagenomics technologies comprehensively revealed the diversity and functions of gut microbiota under health and disease conditions, while culturomics technologies helped isolation and identification of "unculturable" bacteria in the human gut under conventional culture conditions. The combination of these two technologies not only enabled us better understand the FMT regularities of cause and effect in clinical practices, but also effectively promoted its applications. Considering the above advantages, this article summarized the applications of metagenomics and culturomics technologies in FMT and prospected its future development trend.
Humans ; Fecal Microbiota Transplantation ; Metagenomics ; Feces/microbiology* ; Gastrointestinal Microbiome ; Bacteria

Objective: To explore the differences in subsequent analysis between metagenomic and 16Sr DNA sequencing in compositionally characterizing gut microbiota of healthy elderly. Methods: By using a panel study design, five monthly repeated measurements were performed among 76 healthy older people in Jinan City, Shandong Province. Their fecal samples were collected, and genomic DNA was extracted and analyzed through metagenomic and 16Sr DNA sequencing to compare the composition and diversity of gut microbiota. The correlation between species abundance and α diversity was analyzed by Pearson correlation analysis, and the correlation between species abundance and β diversity was determined by Procrustes analysis. Results: The age of 76 participants was (65.07±2.75), and the body mass index was (25.03±2.40) kg/m². There were 38 males and 38 females. A total of 345 fecal samples were obtained from five monthly repeated measurements . Compared with 16S rDNA sequencing, metagenomic sequencing showed more annotated species at each level. The difference in the number of two sequencing species increased with the decrease of the level. Although there were significant differences in species richness between the two sequencing methods. Their species richness was highly correlated at both phylum (r=0.88, P<0.001) and genus (r=0.77, P<0.001) levels. Bacteroidetes and Firmicutes were the common dominant species. Gut microbiota diversity analysis further showed that there was a significantly positive correlation between α diversity (r=0.70, P<0.001) and β diversities (M²=0.84, P<0.05) in the two groups. Conclusion: The annotation efficiency of metagenomic sequencing is much higher than that of 16S rDNA sequencing. The two sequencing methods are consistent in phylum abundance as well as α diversity.
Male ; Female ; Humans ; Aged ; Gastrointestinal Microbiome/genetics* ; DNA, Ribosomal/genetics* ; Feces ; Sequence Analysis, DNA ; Metagenomics ; RNA, Ribosomal, 16S/genetics*

Objective: To explore the value of metagenomic next-generation sequencing (mNGS) in the etiology diagnosis of bacterial meningitis in children. Methods: The etiological results of 189 children diagnosed with "bacterial meningitis" or "purulent meningitis" or "central nervous system infection" in the Children's Hospital of Fudan University from 1st January 2019 to 31st December 2020 were analyzed retrospectively. The cerebrospinal fluid (CFS) of the children with bacterial meningitis was detected by culture and mNGS respectively, and the difference of pathogen detection rate between the 2 methods was analyzed. According to the age at the time of visit, the children were divided into neonatal group (≤28 days of age) and non-neonatal group (>28 days of age), and χ² test was used to compare the positive rate between the 2 groups. Taking CFS culture as the gold standard, the sensitivity and specificity of mNGS in the diagnosing of bacterial meningitis in children were analyzed. Results: Among these 189 children with bacterial meningitis, 116 were males and 73 were females. A total of 76 strains of pathogens were detected in blood and (or) CSF cultures, of which 50 strains (65.8%) were Gram-positive bacteria; among those, 18 strains (23.7%) of Streptococcus agalactiae, 17 strains (19.7%) of Escherichia coli and 15 strains (19.7%) of Streptococcus pneumoniae were detected with higher detection rate. The infection rate of Gram-positive bacteria in the non-neonatal group was higher than that in the neonatal group (76.0% (38/50) vs. 50.0% (13/26), χ²=5.24, P=0.020).The same CSF samples of 48 cases were tested by mNGS and culture at the same time, and the detection rate of mNGS was higher than that of CSF culture (20 cases (41.7%) vs. 12 cases (25.0%), χ²=16.45, P<0.001). The consistency of mNGS and culture results was 79.2% (38/48), and the same pathogen was detected in 11 children with both positive mNGS and CSF culture. Taking the results of CSF culture as the gold standard, the sensitivity of mNGS in the diagnosing of bacterial meningitis was 91.7%, and the specificity was 75.0%. Conclusions: The mNGS technology can improve the pathogen detection rate of bacterial meningitis in children, and has a high consistency with CSF culture. In suspected cases where the pathogen cannot be identified by traditional methods, CSF mNGS should be considered timely.
Child ; Escherichia coli ; Fatigue Syndrome, Chronic ; Female ; Gram-Positive Bacteria ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Male ; Meningitis, Bacterial/microbiology* ; Metagenomics/methods* ; Retrospective Studies ; Sensitivity and Specificity

This paper reported a case of severe Chlamydia psittaci pneumonia. The patient had a clear history of contact with sick poultry. The clinical manifestations were dry cough, fever and respiratory failure. Chest CT showed consolidation in the lower lobe of the right lung, and a small amount of exudative ground-glass opacity in the left lung. Chlamydia psittaci was detected in bronchoalveolar lavage fluid (BALF) by metagenomic assay. After treatment with antibiotics such as nitroimidazoles and carbapenems, the patient was discharged with a better health condition.
Bronchoalveolar Lavage Fluid ; Chlamydophila psittaci ; Humans ; Metagenomics ; Pneumonia ; Psittacosis/drug therapy*

OBJECTIVES: To study the application value of metagenomic next-generation sequencing (mNGS) for pathogen detection in childhood agranulocytosis with fever. METHODS: A retrospective analysis was performed on the mNGS results of pathogen detection of 116 children with agranulocytosis with fever who were treated from January 2020 to December 2021. Among these children, 38 children with negative mNGS results were enrolled as the negative group, and 78 children with positive results were divided into a bacteria group (n=22), a fungal group (n=23), and a viral group (n=31). Clinical data were compared between groups. RESULTS: For the 116 children with agranulocytosis and fever, the median age was 8 years at diagnosis, the median turnaround time of mNGS results was 2 days, and the positive rate of mNGS testing was 67.2% (78/116). Compared with the negative group, the bacterial group had a higher procalcitonin level (P<0.05), the fungal group had higher level of C-reactive protein and positive rate of (1,3)-β-D glucan test/galactomannan test (P<0.05), and the fungal group had a longer duration of fever (P<0.05). Among the 22 positive microbial culture specimens, 9 (41%) were consistent with the mNGS results. Among the 17 positive blood culture specimens, 8 (47%) were consistent with the mNGS results. Treatment was adjusted for 28 children (36%) with the mNGS results, among whom 26 were cured and discharged. CONCLUSIONS The mNGS technique has a shorter turnaround time and a higher sensitivity for pathogen detection and can provide evidence for the pathogenic diagnosis of children with agranulocytosis and fever.
Agranulocytosis/diagnosis* ; Bacteria ; Child ; Fever/diagnosis* ; High-Throughput Nucleotide Sequencing/methods* ; Humans ; Metagenomics/methods* ; Retrospective Studies ; Sensitivity and Specificity