Nanopore sequencing for smear-negative pulmonary tuberculosis-a multicentre prospective study in China.

PURPOSE: In this prospective study, the diagnosis accuracy of nanopore sequencing-based Mycobacterium tuberculosis (MTB) detection was determined through examining bronchoalveolar lavage fluid (BALF) samples from pulmonary tuberculosis (PTB) -suspected patients. Compared the diagnostic performance of nanopore sequencing, mycobacterial growth indicator tube (MGIT) culture and Xpert MTB/rifampin resistance (MTB/RIF) assays. METHODS: Specimens collected from suspected PTB cases across China from September 2021 to April 2022 were tested then assay diagnostic accuracy rates were compared. RESULTS: Among the 111 suspected PTB cases that were ultimately diagnosed as PTB, the diagnostic rate of nanopore sequencing was statistically significant different from other assays (P < 0.05). Fleiss' kappa values of 0.219 and 0.303 indicated fair consistency levels between MTB detection results obtained using nanopore sequencing versus other assays, respectively. Respective PTB diagnostic sensitivity rates of MGIT culture, Xpert MTB/RIF and nanopore sequencing of 36.11%, 40.28% and 83.33% indicated superior sensitivity of nanopore sequencing. Analysis of area under the curve (AUC), Youden's index and accuracy values and the negative predictive value (NPV) indicated superior MTB detection performance for nanopore sequencing (with Xpert MTB/RIF ranking second), while the PTB diagnostic accuracy rate of nanopore sequencing exceeded corresponding rates of the other methods. CONCLUSIONS: In comparison with MGIT culture and Xpert MTB/RIF assays, BALF's nanopore sequencing provided superior MTB detection sensitivity and thus is suitable for testing of sputum-scarce suspected PTB cases. However, negative results obtained using these assays should be confirmed based on additional evidence before ruling out a PTB diagnosis.

Genetic diversity and evolutionary dynamics of Ebola virus in Sierra Leone.

A novel Ebola virus (EBOV) first identified in March 2014 has infected more than 25,000 people in West Africa, resulting in more than 10,000 deaths. Preliminary analyses of genome sequences of 81 EBOV collected from March to June 2014 from Guinea and Sierra Leone suggest that the 2014 EBOV originated from an independent transmission event from its natural reservoir followed by sustained human-to-human infections. It has been reported that the EBOV genome variation might have an effect on the efficacy of sequence-based virus detection and candidate therapeutics. However, only limited viral information has been available since July 2014, when the outbreak entered a rapid growth phase. Here we describe 175 full-length EBOV genome sequences from five severely stricken districts in Sierra Leone from 28 September to 11 November 2014. We found that the 2014 EBOV has become more phylogenetically and genetically diverse from July to November 2014, characterized by the emergence of multiple novel lineages. The substitution rate for the 2014 EBOV was estimated to be 1.23 × 10(-3) substitutions per site per year (95% highest posterior density interval, 1.04 × 10(-3) to 1.41 × 10(-3) substitutions per site per year), approximating to that observed between previous EBOV outbreaks. The sharp increase in genetic diversity of the 2014 EBOV warrants extensive EBOV surveillance in Sierra Leone, Guinea and Liberia to better understand the viral evolution and transmission dynamics of the ongoing outbreak. These data will facilitate the international efforts to develop vaccines and therapeutics.

Single-center study of Lynch syndrome screening in colorectal polyps.

BACKGROUND: Lynch syndrome is the most common hereditary colorectal cancer syndrome, and adenoma is one of the important premalignant lesions to colorectal cancer in Lynch syndrome. The first objective of this study was to calculate the detection rate of Lynch syndrome in colorectal polyps by using mismatch repair immunohistochemistry as the initial screening strategy. The second objective of this study was to optimize screening strategies for adenoma associated with Lynch syndrome by integrating polyp and/or patient characteristics such as polyp size, location, dysplasia, age of onset and/or family history of cancer. METHODS: From June 2014 to May 2016, immunohistochemistry was performed for mismatch repair proteins (MLH1, MSH2, MSH6, and PMS2) using endoscopically resected specimens obtained from newly diagnosed colorectal adenomas. Gene analysis was performed in patients with missing expression of mismatched repair protein. RESULTS: Five hundred and ten patients underwent colorectal polyp resection, with a total of 718 polyps. Five hundred and eight resected adenomas underwent mismatch repair protein immunohistochemical testing. Loss of mismatch repair protein expression was observed in six adenomas, accounting for 1.18% of all adenomas. Five patients then underwent genetic tests to identify two pathogenic mutations from different individuals, while another patient was suspected to have a pathogenic mutation. Three patients were younger than 50 years old. Two patients had advanced histology (high-grade dysplasia and malignant components) and one patient had a family history of cancer. CONCLUSION: Immunohistochemical detection of colorectal polyp mismatch repair protein as Lynch syndrome screening efficiency is low. Effective screening strategies may be improved by optimizing patient/polyp selection, such as focusing on young adenoma patients with a family history of cancer, or patients who present with high-risk features (large size, villous, high-grade dysplasia and malignant components).

[A single center study of colorectal cancer screening for Lynch syndrome].

OBJECTIVE: To determine the ratio of deficient mismatch repair (dMMR) proteins and Lynch syndrome among patients undergoing colorectal cancer resection. METHODS: From June 2014 to May 2016, immunohistochemistry for mismatch repair proteins including mutL homolog 1 (MLH1), mutS homolog 2 (MSH2), mutS homolog 6 (MSH6) and PMS1 homolog 2 (PMS2) were carried out on 207 surgically resected specimens. Samples with lost expression of MMR proteins underwent genetic testing. RESULTS: Loss of expression of MMR proteins were found among 21 patients and accounted for 10.14% of the colorectal cancers. dMMR was more common in patients ≤50 years old, or with proximal tumor at splenic flexure and mucinous adenocarcinoma. Ten patients underwent genetic testing, with three pathogenic mutations (MSH6 c.3013C>T, MLH1 c.199G>A and a novel MSH6 c.584delT) and four ambiguous mutations identified. At least 1.4% of the colorectal cancers were diagnosed as Lynch syndrome. CONCLUSION: Routine screening for Lynch syndrome among patients with colorectal cancer with MMR protein immunohistochemistry as preliminary screening method and MMR gene sequencing as diagnostic method is effective and feasible. It can reduce missed diagnosis of Lynch syndrome and bring lifelong benefit to patients and their families.

Application of next-generation sequencing to characterize novel mutations in clarithromycin-susceptible Helicobacter pylori strains with A2143G of 23S rRNA gene.

BACKGROUND: Clarithromycin (CLR) resistance has become a predominant factor for treatment failure of Helicobacter pylori eradication. Although the molecular mechanism of CLR resistance has been clearly understood in H. pylori, it is lack of evidence of other genes involved in drug resistance. Furthermore, the molecular mechanism of phenotype susceptible to CLR while genotype of 23S rRNA is mutant with A2143G is unclear. Here, we characterized the mutations of CLR-resistant and -susceptible H. pylori strains to explore bacterial resistance. METHODS: In the present study, the whole genomes of twelve clinical isolated H. pylori strains were sequenced, including two CLR-susceptible strains with mutation of A2143G. Single nucleotide variants (SNVs) were extracted and analyzed from multidrug efflux transporter genes. RESULTS: We did not find mutations associated with known CLR-resistant sites except for controversial T2182C outside of A2143G in the 23S rRNA gene. Although total SNVs of multidrug efflux transporter gene and the SNVs of HP0605 were significant differences (P ≤ 0.05) between phenotype resistant and susceptible strains. There is no significant difference in SNVs of RND or MFS (HP1181) family. However, the number of mutations in the RND family was significantly higher in the mutant strain (A2143G) than in the wild type. In addition, three special variations from two membrane proteins of mtrC and hefD were identified in both CLR-susceptible strains with A2143G. CONCLUSIONS: Next-generation sequencing is a practical strategy for analyzing genomic variation associated with antibiotic resistance in H. pylori. The variations of membrane proteins of the RND family may be able to participate in the regulation of clinical isolated H. pylori susceptibility profiles.

The application of targeted nanopore sequencing for the identification of pathogens and resistance genes in lower respiratory tract infections.

Objectives: Lower respiratory tract infections (LRTIs) are one of the causes of mortality among infectious diseases. Microbial cultures commonly used in clinical practice are time-consuming, have poor sensitivity to unculturable and polymicrobial patterns, and are inadequate to guide timely and accurate antibiotic therapy. We investigated the feasibility of targeted nanopore sequencing (TNPseq) for the identification of pathogen and antimicrobial resistance (AMR) genes across suspected patients with LRTIs. TNPseq is a novel approach, which was improved based on nanopore sequencing for the identification of bacterial and fungal infections of clinical relevance. Methods: This prospective study recruited 146 patients suspected of having LRTIs and with a median age of 61 years. The potential pathogens in these patients were detected by both TNPseq and the traditional culture workups. We compared the performance between the two methods among 146 LRTIs-related specimens. AMR genes were also detected by TNPseq to prompt the proper utilization of antibiotics. Results: At least one pathogen was detected in 133 (91.1%) samples by TNPseq, but only 37 (25.3%) samples contained positive isolates among 146 cultured specimens. TNPseq possessed higher sensitivity than the conventional culture method (91.1 vs. 25.3%, P < 0.001) in identifying pathogens. It detected more samples with bacterial infections (P < 0.001) and mixed infections (P < 0.001) compared with the clinical culture tests. The most frequent AMR gene identified by TNPseq was bla TEM (n = 29), followed by bla SHV (n = 4), bla KPC (n = 2), bla CTX-M (n = 2), and mecA (n = 2). Furthermore, TNPseq discovered five possible multi-drug resistance specimens. Conclusion: TNPseq is efficient to identify pathogens early, thus assisting physicians to conduct timely and precise treatment for patients with suspected LRTIs.

Purification, crystallization and preliminary crystallographic analysis of recombinant Lac15 from a marine microbial metagenome.

Laccases are members of the blue multi-copper oxidase family that can oxidize a wide range of aromatic compounds. A new bacterial laccase (Lac15) has recently been obtained from a marine microbial metagenome from the South China Sea and characterized. In this work, recombinant Lac15 was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected to 2.2 Šresolution. The crystal belonged to space group C121, with unit-cell parameters a = 123.41, b = 91.36, c = 86.157 Å, ß = 112.10°.

Integrating nested PCR with high-throughput sequencing to characterize mutations of HBV genome in low viral load samples.

Due to the low viral load of hepatitis B virus (HBV) in plasma samples, conventional techniques have limitations to the detection of antiviral resistance mutations. To solve the problem, we developed a fast, highly sensitive, and accurate method to sequence the HBV whole-genome sequencing in plasma samples which had various viral loads from very low to high.Twenty-one plasma samples were collected from patients who were carriers of HBV from the Hangzhou First People's Hospital. Two pairs of conserved, overlapping, nested primers were used to amplify and sequence the whole HBV genome in 8 plasma samples with different viral loads. High-throughput sequencing was performed on Illumina MiSeq platform. Concomitantly, 3 samples were directly sequenced without PCR amplification. We compared amplicon-sequencing with direct sequencing to develop a method for amplifying and characterizing the whole genome of HBV.HBV genome was amplified from all samples and verified by Sanger sequencing, regardless of the viral loads. Sequencing results revealed that only a few reads were mapped to the HBV genome following direct sequencing, while the amplicon-sequencing reads had a good coverage and depth. We identified 50 intrahost single nucleotide variations (iSNVs), 14 of which were low frequency mutations. Interestingly, iSNVs were more common in low viral load samples than in high viral load samples, and mutations in the reverse transcriptase (RT) region were most prevalent.We conclude that amplicon-sequencing is not only a practical method to detect HBV infection with a high sensitivity and accuracy but also enables to detect mutations in the HBV genome in low viral load samples from HBV-infected patients. Thus, our findings provide a new diagnosis method of HBV infection, which is capable of detection of low frequent mutations in low viral load samples.

Partners of patients with ulcerative colitis exhibit a biologically relevant dysbiosis in fecal microbial metacommunities.

AIM: To investigate alterations in the fecal microbiome using 16S rRNA amplicon sequencing in couples in the same cohabitation environment. METHODS: Fecal samples were collected from eight ulcerative colitis (UC) patients and their healthy partners at Lishui People's Hospital, Zhejiang Province, China. DNA was extracted and the variable regions V3 and V4 of the 16S rRNA genes were PCR amplified using a two-step protocol. Clear reads were clustered into operational taxonomic units (OTUs) at the 97% sequence similarity level using UCLUST v1.2.22. The Wilcoxon rank-sum test (R v3.1.2) was used to compare inter-individual differences. Differences with a P value < 0.05 were considered statistically significant. RESULTS: Fecal microbial communities were more similar among UC patients than their healthy partners (P = 0.024). UC individuals had a lower relative abundance of bacteria belonging to the Firmicutes, especially Blautia, Clostridium, Coprococcus and Roseburia (P < 0.05). Microbiota dysbiosis was detected in UC patients and their healthy partners. Relevant genera included Akkermansiam, Bacteroides, Escherichia, Lactobacillales, Klebsiella and Parabacteroides. The enriched pathways in fecal samples of UC patients were related to lipid and nucleotide metabolism. Additionally, the pathways involved in membrane transport and metabolism of cofactors and vitamins were more abundant in the healthy partners. CONCLUSION: Our results suggested that the microbial composition might be affected in healthy partners cohabiting with UC patients, especially in terms of microbiota dysbiosis.

Analysis of microbial community composition and diversity in postoperative intracranial infection using high­throughput sequencing.

Intracranial infection is one of the most serious complications following neurosurgery. It is well acknowledged that bacteria and fungi are the main pathogens responsible for postoperative intracranial infection. However, the microbial community structure, including composition, abundance and diversity, in postoperative intracranial infection is not fully understood, which greatly compromises our understanding of the necessity and effectiveness of postoperative antibiotic treatment. The present study collected eight cerebrospinal fluid (CSF) samples from patients with intracranial infection following neurosurgical procedures. High­throughput amplicon sequencing for 16S rDNA and internal transcribed spacer (ITS) was performed using the Illumina MiSeq platform to investigate the microbial community composition and diversity between treated and untreated patients. Bioinformatics analysis revealed that the microbial composition and diversity in each patient group (that is, with or without antibiotic treatment) was similar; however, the group receiving antibiotic treatment had a comparatively lower species abundance and diversity compared with untreated patients. At the genus level, Acinetobacter and Staphylococcus were widely distributed in CSF samples from patients with postoperative intracranial infection; in particular, Acinetobacter was detected in all CSF samples. In addition, five ITS fungal libraries were constructed, and Candida was detected in three out of four patients not receiving antibiotic treatment, indicating that the fungal infection should be given more attention. In summary, 16S and ITS high­throughput amplicon sequencing were practical methods to identify pathogens in the different periods of treatment in patients with postoperative intracranial infection. There was a notable difference in microbial composition and diversity between the treated and untreated patients. Alterations in the microbial community structure may provide a signal whether antibiotic treatment worked in postoperative intracranial infection and may assist surgeons to better control the progression of infection.

Direct next-generation sequencing of virus-human mixed samples without pretreatment is favorable to recover virus genome.

Next-generation sequencing (NGS) enables the recovery of pathogen genomes from clinical samples without the need for culturing. Depletion of host/microbiota components (e.g., ribosomal RNA and poly-A RNA) and whole DNA/cDNA amplification are routine methods to improve recovery results. Using mixtures of human and influenza A virus (H1N1) RNA as a model, we found that background depletion and whole transcriptome amplification introduced biased distributions of read coverage over the H1N1 genome, thereby hampering genome assembly. Influenza serotyping was also affected by pretreatments. We propose that direct sequencing of noncultured samples without pretreatment is a favorable option for pathogen genome recovery applications.

Intra-host dynamics of Ebola virus during 2014.

Since 2013, West Africa has encountered the largest Ebola virus (EBOV) disease outbreak on record, and Sierra Leone is the worst-affected country, with nearly half of the infections. By means of next-generation sequencing and phylogeographic analysis, the epidemiology and transmission of EBOV have been well elucidated. However, the intra-host dynamics that mainly reflect viral-host interactions still need to be studied. Here, we show a total of 710 intra-host single nucleotide variations (iSNVs) from deep-sequenced samples from EBOV-infected patients, through a well-tailored bioinformatics pipeline. We present a comprehensive distribution of iSNVs during this outbreak and along the EBOV genome. Analyses of iSNV and its allele frequency reveal that VP40 is the most conserved gene during this outbreak, and thus it would be an ideal therapeutic target. In the co-occurring iSNV network, varied iSNV sites present different selection features. Intriguingly, the T-to-C substitutions at the 3'-UTR of the nucleoprotein (NP; positions 3008 and 3011), observed in many patients, result in the upregulation of the transcription of NP through an Ebola mini-genome reporting system. Additionally, no iSNV enrichment within B-cell epitopes of GP has been observed.

Metagenomic sequencing of bile from gallstone patients to identify different microbial community patterns and novel biliary bacteria.

Despite the high worldwide prevalence of gallstone disease, the role of the biliary microbiota in gallstone pathogenesis remains obscure. Next-generation sequencing offers advantages for systematically understanding the human microbiota; however, there have been few such investigations of the biliary microbiome. Here, we performed whole-metagenome shotgun (WMS) sequencing and 16S rRNA sequencing on bile samples from 15 Chinese patients with gallstone disease. Microbial communities of most individuals were clustered into two types, according to the relative enrichment of different intestinal bacterial species. In the bile samples, oral cavity/respiratory tract inhabitants were more prevalent than intestinal inhabitants and existed in both community types. Unexpectedly, the two types were not associated with fever status or surgical history, and many bacteria were patient-specific. We identified 13 novel biliary bacteria based on WMS sequencing, as well as genes encoding putative proteins related to gallstone formation and bile resistance (e.g., ß-glucuronidase and multidrug efflux pumps). Bile samples from gallstone patients had reduced microbial diversity compared to healthy faecal samples. Patient samples were enriched in pathways related to oxidative stress and flagellar assembly, whereas carbohydrate metabolic pathways showed varying behaviours. As the first biliary WMS survey, our study reveals the complexity and specificity of biliary microecology.