30 research outputs found
Metagenomic analysis of colonic tissue and stool microbiome in patients with colorectal cancer in a South Asian population
No full textBackground: The gut microbiome is thought to play an important role in the development of colorectal cancer (CRC). However, as the gut microbiome varies widely based on diet, we sought to investigate the gut microbiome changes in patients with CRC in a South Asian population. Methods: The gut microbiome was assessed by 16s metagenomic sequencing targeting the V4 hypervariable region of the bacterial 16S rRNA in stool samples (n = 112) and colonic tissue (n = 36) in 112 individuals. The cohort comprised of individuals with CRC (n = 24), premalignant lesions (n = 10), healthy individuals (n = 50) and in those with diabetes (n = 28). Results: Overall, the relative abundances of genus Fusobacterium (p < 0.001), Acinetobacter (p < 0.001), Escherichia-Shigella (p < 0.05) were significantly higher in gut tissue, while Romboutsia (p < 0.01) and Prevotella (p < 0.05) were significantly higher in stool samples. Bacteroides and Fusobacterium were the most abundant genera found in stool samples in patients with CRC. Patients with pre-malignant lesions had significantly high abundances of Christensenellaceae, Enterobacteriaceae, Mollicutes and Ruminococcaceae (p < 0.001) compared to patients with CRC, and healthy individuals. Romboutsia was significantly more abundant (p < 0.01) in stool samples in healthy individuals compared to those with CRC and diabetes. Conclusion: Despite marked differences in the Sri Lankan diet compared to the typical Western diet, Bacteroides and Fusobacterium species were the most abundant in those with CRC, with Prevotella species, being most abundant in many individuals. We believe these results pave the way for possible dietary interventions for prevention of CRC in the South Asian population
Proteomics Analysis of Peripheral Blood Mononuclear Cells from Patients in Early Dengue Infection Reveals Potential Markers of Subsequent Fluid Leakage
No full textInfections caused by dengue virus (DENV) result in significant morbidity and mortality. A proportion of infected individuals develop dengue haemorrhagic fever (DHF) characterized by circulatory collapse and multiorgan failure. Early detection of individuals likely to develop DHF could lead to improved outcomes for patients and help us use healthcare resources more efficiently. We identified proteins that are differentially regulated during early disease in peripheral blood mononuclear cells (PBMCs) of patients who subsequently developed DHF. Four dengue fever (DF), four DHF and two healthy control PBMCs were subjected to tandem mass tag mass spectrometry. Differentially regulated proteins were used to identify up- or down-regulated Gene Ontology pathways. One hundred and sixty proteins were differentially expressed in DENV-infected samples compared to healthy controls. PBMCs from DHF patients differentially expressed 90 proteins compared to DF; these were involved in down-regulation of platelet activation and aggregation, cell adhesion, and cytoskeleton arrangement pathways. Proteins involved in oxidative stress and p38 MAPK signalling were upregulated in DHF samples during early infection compared to DF. This study has identified 90 proteins differentially regulated in PBMCs that could potentially serve as biomarkers to identify patients at risk of developing DHF at an early disease stage
Regional Variation in Dengue Virus Serotypes in Sri Lanka and Its Clinical and Epidemiological Relevance
No full textDengue is a significant health concern in Sri Lanka, but diagnosis of the infecting dengue virus (DENV) serotype has hitherto been largely restricted to the Colombo district in the western province. Salinity tolerant Aedes vectors are present in the island’s northern Jaffna peninsula, which is undergoing rapid groundwater salinization. Virus serotypes were determined by RT-qPCR in 107 and 112 patients diagnosed by NS1 antigen positivity from the Jaffna district in 2018 and 2019, respectively, and related to clinical characteristics. DENV1 and DENV2 were the most common serotypes in both years. Infections with multiple serotypes were not detected. DENV1 was significantly more prevalent in 2019 than 2018, while DENV3 was significantly more prevalent in 2018 than 2019 among the Jaffna patients. Limited genomic sequencing identified DENV1 genotype-I and DENV3 genotype-I in Jaffna patients in 2018. Dengue was more prevalent in working age persons and males among the serotyped Jaffna patients. DENV1 and DENV2 were the predominant serotypes in 2019 in the Colombo district. However, DENV1 and DENV3 were significantly more prevalent in Colombo compared with Jaffna in 2019. The differences in the prevalence of DENV1 and DENV3 between the Jaffna and Colombo districts in 2019 have implications for dengue epidemiology and vaccination. Salinity-tolerant Aedes vector strains, widespread in the Jaffna peninsula, may have contributed to differences in serotype prevalence compared with the Colombo district in 2019. Significant associations were not identified between virus serotypes and clinical characteristics among Jaffna patients
Genomic analysis of a novel Rhodococcus (Prescottella) equi isolate from a bovine host
No full textRhodococcus (Prescottella) equi causes pneumonia-like infections in foals with high mortality rates and can also infect a number of other animals. R. equi is also emerging as an opportunistic human pathogen. In this study, we have sequenced the genome of a novel R. equi isolate, B0269, isolated from the faeces of a bovine host. Comparative genomic analyses with seven other published R. equi genomes, including those from equine or human sources, revealed a pangenome comprising of 6876 genes with 4141 genes in the core genome. Two hundred and 75 genes were specific to the bovine isolate, mostly encoding hypothetical proteins of unknown function. However, these genes include four copies of terA and five copies of terD genes that may be involved in responding to chemical stress. Virulence characteristics in R. equi are associated with the presence of large plasmids carrying a pathogenicity island, including genes from the vap multigene family. A BLAST search of the protein sequences from known virulence-associated plasmids (pVAPA, pVAPB and pVAPN) revealed a similar plasmid backbone on two contigs in bovine isolate B0269; however, no homologues of the main virulence-associated genes, vapA, vapB or vapN, were identified. In summary, this study confirms that R. equi genomes are highly conserved and reports the presence of an apparently novel plasmid in the bovine isolate B0269 that needs further characterisation to understand its potential involvement in virulence properties
Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR.
No full textAs different SARS-CoV-2 variants emerge and with the continuous evolvement of sub lineages of the delta variant, it is crucial that all countries carry out sequencing of at least >1% of their infections, in order to detect emergence of variants with higher transmissibility and with ability to evade immunity. However, due to limited resources as many resource poor countries are unable to sequence adequate number of viruses, we compared to usefulness of a two-step commercially available multiplex real-time PCR assay to detect important single nucleotide polymorphisms (SNPs) associated with the variants and compared the sensitivity, accuracy and cost effectiveness of the Illumina sequencing platform and the Oxford Nanopore Technologies' (ONT) platform. 138/143 (96.5%) identified as the alpha and 36/39 (92.3%) samples identified as the delta variants due to the presence of lineage defining SNPs by the multiplex real time PCR, were assigned to the same lineage by either of the two sequencing platforms. 34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using Illumina generated <5%. However, the mean PHRED scores averaged at 32.35 by Illumina reads but 10.78 in ONT. This difference results in a base error probability of 1 in 10 by the ONT and 1 in 1000 for Illumina sequencing platform. Sub-consensus single nucleotide variations (SNV) are highly correlated between both platforms (R2 = 0.79) while indels appear to have a weaker correlation (R2 = 0.13). Although the ONT had a slightly higher error rate compared to the Illumina technology, it achieved higher coverage with a lower number or reads, generated less ambiguous bases and was significantly less expensive than Illumina sequencing technology
Correlation of sub-consensus allele frequencies observed for SNV and Indels between two sequencing technologies.
No full texta) Correlation between sub-consensus single nucleotide substitution frequencies observed for Illumina and ONT. Nucleotide substitutions detected exclusively by one technology are indicated in green and blue whereas the substitutions detected by both technologies are colored red. Even though more nucleotide substitutions exclusive to ONT were observed, there is a clear positive correlation (R2 = 0.79) between two sequencing technologies. b) Correlation between sub-consensus indel frequencies observed for Illumina and ONT. More indels exclusive to ONT can be seen with a weak correlation (R2 = 0.13) between the indel frequencies between two technologies suggesting ONT tend to result in more false-positive indels.</p
Basic sequencing matrices for Illumina and Oxford Nanopore (ONT) outputs of 37.
No full textBasic sequencing matrices for Illumina and Oxford Nanopore (ONT) outputs of 37.</p
PHRED base call quality score distribution of samples sequenced by Illumina and ONT.
No full textDistribution plot of PHRED (probability of error per base call in a log scale) quality score (x axis) and error probability (secondary x axis) derived from the PHRED score for the data set sequenced from Illumina (n = 37) and ONT (n = 37). The scores of ONT are shown in blue and Illumina in red. The mean PHRED scores/error probability are shown with the dashed line for each technology. The mean PHRED scores averaged at 32.35 in Illumina reads and 10.78 in ONT.</p
Combined maximum likelihood phylogenetic tree created using sequence pairs of 37 the samples.
No full textThe ML tree was generated using the consensus sequences of each sequencing technology with 1000 bootstrap replicates using TIM2+F+R2 model. Tree is rooted on SARS-CoV-2 reference MN908947.3 and with samples sequences by Illumina coloured red and those sequenced by ONT coloured blue. Bootstrap support values are shown on each branch. 21/37 samples coupled together with 90% genome coverage from both Illumina and ONT datasets while 7/37 samples coupled together with less than 98% bootstrap support. 9/37 of the samples which failed to couple with their counterpart from ONT or Illumina had moderate to high (3% - 31%) ambiguous bases in either sequences.</p
Molecular epidemiology and evolutionary trends of dengue virus serotype-2 strains in Sri Lanka
No full textBackground: Dengue virus (DENV) infections in Sri Lanka have intensified over the past 3 decades. Surveillance and characterization of different DENV lineages of different serotypes causing outbreaks is crucial for initiation of timely dengue control measures and for implementing dengue vaccines. Therefore, we characterized the DENV-2 strains in Sri Lanka from 2016, until end of 2023 and their evolutionary dynamics to understand the geographical spread and mutations arising with the DENV-2 serotypes in Sri Lanka. Methodology: Sequencing was carried out on 80 DENV-2 samples collected from patients with acute dengue recruited in the years 2016 to 2018, and on 12 DENV-2 samples in patients recruited in years 2022 to 2023 using Oxford Nanopore Technology. Phylogenetic analysis was carried out using the IQ-TREE tool, and Galaxy to construct a phylogenetic tree from the aligned sequence data. The sequences were also analyzed for non-synonymous amino acid changes in the envelope and NS1 regions. Results: The Sri Lankan DENV-2 sequences circulating from 2016 to end of 2023 belonged to genotype II.F.1.1 lineage. They were closely related to strains circulating in the same period in South Asia and Southeast Asia. We identified 15 non-synonymous mutations within the envelope region and 22 non-synonymous mutations within the NS1 region, with 7 non-synonymous mutations within the E region (M6I, Q52H, E71A, V129I, N390S, I484V, T478S) and 10 non-synonymous mutations within the NS1 region (S80T, T117A, Q131H, K174R, F178S, N222S, L247F, I264T, T265A, K272R) seen in all sequenced samples. Some of these mutations were previously shown to be associated with increase in viral replication, NS1 secretion and immune evasion, while some have not been reported elsewhere. Conclusions: Given the increase in dengue transmission in many countries, it is important to further strengthen DENV surveillance for studying the evolutionary patterns of the DENV to initiate timely and appropriate control measures
