Investigation of neglected protists Blastocystis sp. and Dientamoeba fragilis in immunocompetent and immunodeficient diarrheal patients using both conventional and molecular methods

Introduction The clinical significance of Blastocystis sp. and Dientamoeba fragilis in patients with gastrointestinal symptoms is a controversial issue. Since the pathogenicity of these protists has not been fully elucidated, testing for these organisms is not routinely pursued by most laboratories and clinicians. Thus, the prevalence of these organisms and the subtypes of Blastocystis sp. in human patients in Turkey are not well characterized. This study aimed to determine the prevalence of Blastocystis sp. and D. fragilis in the diarrheic stool samples of immunodeficient and immunocompetent patients using conventional and molecular methods and to identify Blastocystis sp. subtypes using next generation sequencing. Material and methods Individual stool specimens were collected from 245 immunodeficient and 193 immunocompetent diarrheic patients between March 2017 and December 2019 at the Gazi University Training and Research Hospital in Ankara, Turkey. Samples were screened for Blastocystis sp. and D. fragilis by conventional and molecular methods. Molecular detection of both protists was achieved by separate qPCRs targeting a partial fragment of the SSU rRNA gene. Next generation sequencing was used to identify Blastocystis sp. subtypes. Results The prevalence of Blastocystis sp. and D. fragilis was 16.7% and 11.9%, respectively as measured by qPCR. The prevalence of Blastocystis sp. and D. fragilis was lower in immunodeficient patients (12.7% and 10.6%, respectively) compared to immunocompetent patients (21.8% and 13.5%, respectively). Five Blastocystis sp. subtypes were identified and the following subtype distribution was observed: ST3 54.4% (n = 37), ST2 16.2% (n = 11), ST1 4.4% (n = 3), ST6 2.9% (n = 2), ST4 1.5% (n = 1), ST2/ST3 11.8% (n = 8) and ST1/ST3 8.8% (n = 6). There was no statistically significant difference in the distribution of Blastocystis sp. subtypes between immunocompetent and immunodeficient patients. Conclusion and recommendation Our findings demonstrated that Blastocystis sp. and D. fragilis are commonly present in immunocompetent and immunodeficient patients with diarrhea. This study is the first to use next generation sequencing to address the presence of Blastocystis sp. mixed subtypes and intra-subtype variability in clinical samples in Turkey.

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Evaluation of the Use of Formalin-Fixed and Paraffin-Embedded Archive Gastric Tissues for Microbiota Characterization Using Next-Generation Sequencing

International Journal of Molecular Sciences ◽

10.3390/ijms21031096 ◽

2020 ◽

Vol 21 (3) ◽

pp. 1096 ◽

Cited By ~ 1

Author(s):

Ines Pinto-Ribeiro ◽

Rui M. Ferreira ◽

Joana Pereira-Marques ◽

Vanessa Pinto ◽

Guilherme Macedo ◽

...

Keyword(s):

Next Generation Sequencing ◽

Tissue Sample ◽

Clinical Samples ◽

Rrna Gene ◽

Next Generation ◽

Sequencing Data ◽

Ffpe Tissues ◽

The Times ◽

Generation Sequencing ◽

Formalin Fixed

Large numbers of well-characterized clinical samples are fundamental to establish relevant associations between the microbiota and disease. Formalin-fixed and paraffin-embedded (FFPE) tissues are routinely used and are widely available clinical materials. Since current approaches to study the microbiota are based on next-generation sequencing (NGS) targeting the bacterial 16S rRNA gene, our aim was to evaluate the feasibility of FFPE gastric tissues for NGS-based microbiota characterization. Analysis of sequencing data revealed the presence of bacteria in the paraffin control. After the subtraction of the operational taxonomic units (OTUs) present in the paraffin control to the FFPE tissue sample dataset, we evaluated the microbiota profiles between paired FFPE and frozen gastric tissues, and between different times of archiving. Compared with frozen gastric tissues, we detected a lower number of OTUs in the microbiota of paired FFPE tissues, regardless of the time of archiving. No major differences in microbial diversity were identified, but taxonomic variation in the relative abundance of phyla and orders was observed between the two preservation methods. This variation was also evident in each case and throughout the times of FFPE archiving. The use of FFPE tissues for NGS-based microbiota characterization should be considered carefully, as biases can be introduced by the embedding process and the time of tissue archiving.

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Metagenomic Next-Generation Sequencing for Pathogenic Diagnosis and Antibiotic Management of Severe Community-Acquired Pneumonia in Immunocompromised Adults

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.661589 ◽

2021 ◽

Vol 11 ◽

Author(s):

Ting Sun ◽

Xiaojing Wu ◽

Ying Cai ◽

Tianshu Zhai ◽

Linna Huang ◽

...

Keyword(s):

Next Generation Sequencing ◽

Antibiotic Treatment ◽

Community Acquired Pneumonia ◽

Immunocompromised Patients ◽

Next Generation ◽

Days Of Therapy ◽

Significant Difference ◽

Severe Community Acquired Pneumonia ◽

Generation Sequencing ◽

Immunocompetent Patients

BackgroundMetagenomic next-generation sequencing (mNGS) is a promising technique for pathogens diagnosis. However, application of mNGS in immunocompromised adults with severe community-acquired pneumonia (SCAP) is relatively limited.MethodsWe retrospectively reviewed 23 immunocompromised and 21 immunocompetent SCAP patients with mNGS detection from April 2019 to December 2019. The performances of pathogenic diagnosis and subsequently antibiotic adjustment in immunocompromised SCAP patients were compared to immunocompetent SCAP patients. The defined by days of therapy (DOT) method was used for estimate daily antibiotic use.ResultsThere was a significant difference in the diagnostic positivity rate between mNGS and conventional test in both groups (P<0.001). Compared to immunocompetent patients, more mixed pathogens in immunocompromised patients were found (P=0.023). Before the availability of mNGS, the DOTs in immunocompromise patients were higher than immunocompetent patients (3.0 [3.0, 4.0] vs. 3.0 [2.0, 3.0], P=0.013). Compared to immunocompetent patients, immunocompromised patients had fewer full pathogen covered empirical antibiotic therapy (14.7% vs. 57.1%, P=0.022), more adjustments of antibiotic treatment (87.0%) vs. 57.1%, P=0.027). More than a half (13 of 23) SCAP patients in immunosuppressed group had reduced or downgraded antibiotic adjustments based on the results.ConclusionsmNGS may be a useful technique for detecting mixed pathogens and personalized antibiotic treatment in immunocompromised SCAP patients.

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Utilizing next-generation sequencing to identify prey DNA in western North Atlantic grey seal Halichoerus grypus diet

Marine Ecology Progress Series ◽

10.3354/meps13520 ◽

2020 ◽

Vol 655 ◽

pp. 227-240

Author(s):

KR Flanders ◽

ZH Olson ◽

KA Ono

Keyword(s):

Next Generation Sequencing ◽

Feeding Habits ◽

Atlantic Menhaden ◽

Halichoerus Grypus ◽

Frequency Of Occurrence ◽

Grey Seal ◽

Next Generation ◽

Significant Difference ◽

Generation Sequencing ◽

Seal Diet

Increasing grey seal Halichoerus grypus abundance in coastal New England is leading to social, political, economic, and ecological controversies. Central to these issues is the foraging ecology and diet composition of the seals. We studied grey seal feeding habits through next-generation sequencing of prey DNA using 16S amplicons from seal scat (n = 74) collected from a breeding colony on Monomoy Island in Massachusetts, USA, and report frequency of occurrence and relative read abundance. We also assigned seal sex to scat samples using a revised PCR assay. In contrast to current understanding of grey seal diet from hard parts and fatty acid analysis, we found no significant difference between male and female diet measured by alpha and beta diversity. Overall, we detected 24 prey groups, 18 of which resolved to species. Sand lance Ammodytes spp. were the most frequently consumed prey group, with a frequency of occurrence (FO) of 97.3%, consistent with previous studies, but Atlantic menhaden Brevoortia tyrannus, the second most frequently consumed species (FO = 60.8%), has not previously been documented in US grey seal diet. Our results suggest that a metabarcoding approach to seal food habits can yield important new ecological insights, but that traditional hard parts analysis does not underestimate consumption of Atlantic cod Gadus morhua (FO = 6.7%, Gadidae spp.) and salmon Salmo salar (FO = 0%), 2 particularly valuable species of concern.

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Data analysis workflow for the detection of canine vector-borne pathogens using 16 S rRNA Next-Generation Sequencing

BMC Veterinary Research ◽

10.1186/s12917-021-02969-9 ◽

2021 ◽

Vol 17 (1) ◽

Author(s):

Elton J. R. Vasconcelos ◽

Chayan Roy ◽

Joseph A. Geiger ◽

Kristina M. Oney ◽

Melody Koo ◽

...

Keyword(s):

Veterinary Medicine ◽

Next Generation Sequencing ◽

Complete Analysis ◽

Molecular Diagnostic ◽

Rrna Gene ◽

Spotted Fever Group ◽

Next Generation ◽

Vector Borne ◽

16 S Rrna ◽

Generation Sequencing

Abstract Background Vector-borne diseases (VBDs) impact both human and veterinary medicine and pose special public health challenges. The main bacterial vector-borne pathogens (VBPs) of importance in veterinary medicine include Anaplasma spp., Bartonella spp., Ehrlichia spp., and Spotted Fever Group Rickettsia. Taxon-targeted PCR assays are the current gold standard for VBP diagnostics but limitations on the detection of genetically diverse organisms support a novel approach for broader detection of VBPs. We present a methodology for genetic characterization of VBPs using Next-Generation Sequencing (NGS) and computational approaches. A major advantage of NGS is the ability to detect multiple organisms present in the same clinical sample in an unsupervised (i.e. non-targeted) and semi-quantitative way. The Standard Operating Procedure (SOP) presented here combines industry-standard microbiome analysis tools with our ad-hoc bioinformatic scripts to form a complete analysis pipeline accessible to veterinary scientists and freely available for download and use at https://github.com/eltonjrv/microbiome.westernu/tree/SOP. Results We tested and validated our SOP by mimicking single, double, and triple infections in genomic canine DNA using serial dilutions of plasmids containing the entire 16 S rRNA gene sequence of (A) phagocytophilum, (B) v. berkhoffii, and E. canis. NGS with broad-range 16 S rRNA primers followed by our bioinformatics SOP was capable of detecting these pathogens in biological replicates of different dilutions. These results illustrate the ability of NGS to detect and genetically characterize multi-infections with different amounts of pathogens in a single sample. Conclusions Bloodborne microbiomics & metagenomics approaches may help expand the molecular diagnostic toolbox in veterinary and human medicine. In this paper, we present both in vitro and in silico detailed protocols that can be combined into a single workflow that may provide a significant improvement in VBP diagnostics and also facilitate future applications of microbiome research in veterinary medicine.

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Targeted Next-Generation Sequencing and Informatics as an Effective Tool to Establish the Composition of Bovine Piroplasm Populations in Endemic Regions

Microorganisms ◽

10.3390/microorganisms9010021 ◽

2020 ◽

Vol 9 (1) ◽

pp. 21

Author(s):

Abdul Ghafar ◽

Anson V. Koehler ◽

Ross S. Hall ◽

Charles G. Gauci ◽

Robin B. Gasser ◽

...

Keyword(s):

Next Generation Sequencing ◽

Water Buffalo ◽

Hypervariable Region ◽

Small Subunit ◽

Rrna Gene ◽

Next Generation ◽

Small Subunit Rrna ◽

Tropical Theileriosis ◽

Ecological Zones ◽

Generation Sequencing

Protists of the genera Babesia and Theileria (piroplasms) cause some of the most prevalent and debilitating diseases for bovines worldwide. In this study, we established and used a next-generation sequencing-informatic approach to explore the composition of Babesia and Theileria populations in cattle and water buffalo in a country (Pakistan) endemic for these pathogens. We collected individual blood samples from cattle (n = 212) and water buffalo (n = 154), extracted genomic DNAs, PCR-amplified the V4 hypervariable region of 18S small subunit rRNA gene from piroplasms, sequenced amplicons using Illumina technology, and then analysed data using bioinformatic platforms. The results revealed piroplasms in 68.9% (252/366) samples, with overall occurrence being markedly higher in cattle (85.8%) than in water buffaloes (45.5%). Babesia (B.) occultans and Theileria (T.) lestoquardi-like species were recorded for the first time in Pakistan, and, overall, T. annulata was most commonly detected (65.8%) followed by B. bovis (7.1%), B. bigemina (4.4%), and T. orientalis (0.5%), with the genetic variability within B. bovis being pronounced. The occurrence and composition of piroplasm species varied markedly across different agro-ecological zones. The high detection of T. annulata in asymptomatic animals suggested a relatively high level of endemic stability of tropical theileriosis in the bovine population.

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Next-Generation Sequencing for Pathogen Identification in Infected Foot Ulcers

Foot & Ankle Orthopaedics ◽

10.1177/2473011420s00027 ◽

2020 ◽

Vol 5 (4) ◽

pp. 2473011420S0002

Author(s):

Yoonjung Choi ◽

Irvin Oh

Keyword(s):

Next Generation Sequencing ◽

False Positive ◽

Bacterial Genome ◽

Rrna Gene ◽

Next Generation ◽

Antibiotic Resistant ◽

Streptococcus Species ◽

Resistant Genes ◽

Standard Culture ◽

Generation Sequencing

Category: Other Introduction/Purpose: Foot infections are often polymicrobial with diverse microbiomes. Accurate identification of the main pathogen in diabetic foot ulcer (DFU) remain challenging due to contamination or negative cultures often leading to ineffective post-surgical antibiotic treatment. Application of molecular diagnostics, such as next generation sequencing (NGS) has been explored as an alternative to standard culture in orthopaedic infections. NGS is highly sensitive and detects an entire bacterial genome along with pharmacologic resistant genes in a given sample. We sought to investigate the potential use of NGS for accurate diagnosis and quantification of various species in infected DFU. We hypothesize that NGS will provide a more accurate means of diagnosing and profiling microorganisms in infected DFU compared to the standard culture method. Methods: We investigated 30 infected DFU patients who underwent surgical treatment by a single academic orthopaedic surgeon from October 2018 to September 2019. The average age of the patient was 60.4 (range 33-82) years-old. Surgical procedures performed were irrigation and debridement (12), toe or ray amputation (13), calcanectomies (4), and below-knee amputation (1). Infected bone specimens were obtained intraoperatively and processed for standard culture and NGS. Quantitative PCR was performed to determine the bacterial burden present in the sample. DNA was amplified by PCR from a highly conserved region of the rRNA gene in the bacteria (16S rRNA). Once a high level of DNA was generated and determined, it was compared against NIH GenBank database. Concordance between the standard culture and NGS was assessed. Results: In 28 of 29 patients, pathogens were identified by both NGS and culture, with complete consistency of organisms in 13 cases (concordance rate: 43.3%). NGS provided relative quantitative measures and the presence of antibiotic resistant genes for each pathogen. In NGS, Anaerococcus species (79.3%) was the most common organism, followed by Streptococcus species (44.8%), Prevotella species (44.8%), Finegoldia magna (44.8%). In culture, S. aureus (58.6%) was the most common, followed by Streptococcus species (34.5%), coagulase-negative Staphylococci (24.1%), Corynebacterium species (20.7%). On average, NGS revealed 5.1 (1-11) number of pathogens, whereas standard culture revealed 2.6 (1-6) pathogens in a given sample. NGS identified 2 cases with false positive standard culture and detected antibiotic resistant organisms in 15 specimens. Conclusion: NGS is an emerging method of microbial identification in orthopedic infection. It is particularly helpful in profiling diverse microbes in polymicrobial infected DFU. It can identify major pathogens and may correct false positive or false negative culture. NGS may allow a faster invitation of postoperative targeted antibiotic therapy. [Table: see text]

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The genetic analysis of Chinese patients with clonal cytopenias using targeted next-generation sequencing

Molecular Cytogenetics ◽

10.1186/s13039-021-00572-z ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Lijuan Zhang ◽

YuYe Shi ◽

Yue Chen ◽

Shandong Tao ◽

Wenting Shi ◽

...

Keyword(s):

Next Generation Sequencing ◽

Myelodysplastic Syndromes ◽

Rna Splicing ◽

Myeloproliferative Neoplasms ◽

Chinese Patients ◽

Newly Diagnosed ◽

Next Generation ◽

Targeted Next Generation Sequencing ◽

Significant Difference ◽

Generation Sequencing

Abstract Background Clonal hematopoiesis (CH) can be found in various myeloid neoplasms (MN), such as myelodysplastic syndromes (MDS), myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN), also in pre-MDS conditions. Methods Cytogenetics is an independent prognostic factor in MDS, and fluorescence in-situ hybridization (FISH) can be used as an adjunct to karyotype analysis. In the past 5 years, only 35 of 100 newly diagnosed MDS and MDS/MPN patients were identified abnormalities, who underwent the FISH panel. In addition, we examined a cohort of 51 cytopenic patients suspected MDS or MDS/MPN with a 20-gene next generation sequencing (NGS), including 35 newly diagnosed MN patients and 16 clonal cytopenias of undetermined significance (CCUS) patients. Results Compared with the CCUS group, the MN group had higher male ratio (22/13 vs 10/6), cytogenetics abnormalities rate (41.4% vs 21.4%) and frequency of a series of mutations, such as ASXL1 (28.6% vs 25%), U2AF1 (25.7% vs 25%), RUNX1 (20% vs 0.0%); also, higher adverse mutations proportion (75% vs 85.2%), and double or multiple mutations (54.3% vs 43.75%). There were 7 MN patients and 4 CCUS patients who experienced cardio-cerebrovascular embolism events demonstrated a significant difference between the two groups (25% vs 20%). Ten of the 11 patients had somatic mutations, half had DNA methylation, while the other half had RNA splicing. Additionally, six patients had disease transformation, and four patients had mutated U2AF1, including two CCUS cases and two MDS-EB cases. Following up to January 2021, there was no significant difference in over survival between the CCUS and MN groups. Conclusion NGS facilitates the diagnosis of unexplained cytopenias. The monitoring and management of CCUS is necessary, also cardio-cerebrovascular embolism events in patients with CH need attention in the clinical practice.

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Serra da Estrela PDO Cheese Microbiome as Revealed by Next Generation Sequencing

Microorganisms ◽

10.3390/microorganisms9102007 ◽

2021 ◽

Vol 9 (10) ◽

pp. 2007

Author(s):

Rui Rocha ◽

Manuela Vaz Velho ◽

Joana Santos ◽

Paulo Fernandes

Keyword(s):

Next Generation Sequencing ◽

Relative Abundance ◽

Raw Materials ◽

Rrna Gene ◽

Next Generation ◽

Candida Spp ◽

Characterization Study ◽

Materials Used ◽

Serra Da Estrela ◽

Generation Sequencing

Serra da Estrela PDO cheese is the oldest traditional cheese manufactured in Portugal. In this work, its microbiome as well as the main raw materials used in cheese production, raw ewes’ milk and thistle flowers (Cynara cardunculus L.), were characterized using next generation sequencing. Samples were accordingly retrieved from a local producer over two consecutive production campaigns and at different time periods within each campaign. The bacterial and fungi communities associated with each matrix were accessed through sequencing of V3−V4 and Internal Transcribed Spacer 2 regions of rRNA gene amplicons, respectively. A high microbial diversity was found associated to each matrix, differing significantly (p < 0.05) from each other. Over 500 taxa were identified in each analyzed matrix, ranging from dominant (relative abundance > 1%), sub-dominant (0.01−1%) and rare taxa (<0.01%). Specifically, in cheese, 30 taxa were present in all analyzed samples (core taxa), including species of Leuconostoc spp. and Lactococcus spp. for bacteria and Candida spp., Debaryomyces spp. and Yarrowia spp. for fungi, that were cumulatively the most prevalent genera in Serra da Estrela PDO cheese (average relative abundance ≥10%). Ultimately, this characterization study may contribute to a better understanding of the microbial dynamics of this traditional PDO product, namely the influence of raw materials on cheese microbiome, and could assist producers interested in preserving the identity, quality and safety of Serra da Estrela PDO cheese.

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Microsatellite instability detection using a large next-generation sequencing cancer panel across diverse tumour types

Journal of Clinical Pathology ◽

10.1136/jclinpath-2019-206136 ◽

2019 ◽

Vol 73 (2) ◽

pp. 83-89 ◽

Cited By ~ 6

Author(s):

Jiuhong Pang ◽

Tatyana Gindin ◽

Mahesh Mansukhani ◽

Helen Fernandes ◽

Susan Hsiao

Keyword(s):

Next Generation Sequencing ◽

Microsatellite Instability ◽

Microsatellite Loci ◽

Clinical Samples ◽

Next Generation ◽

Diverse Range ◽

Tree Classifier ◽

Generation Sequencing ◽

Cancer Panel ◽

Clinical Cases

AimMicrosatellite instability (MSI), a hallmark of DNA mismatch repair deficiency, is a key molecular biomarker with multiple clinical implications including the selection of patients for immunotherapy, identifying patients who may have Lynch syndrome and predicting prognosis in patients with colorectal tumours. Next-generation sequencing (NGS) provides the opportunity to interrogate large numbers of microsatellite loci concurrently with genomic variants. We sought to develop a method to detect MSI that would not require paired normal tissue and would leverage the sequence data obtained from a broad range of tumours tested using our 467-gene NGS Columbia Combined Cancer Panel (CCCP).MethodsAltered mononucleotide and dinucleotide microsatellite loci across the CCCP region of interest were evaluated in clinical samples encompassing a diverse range of tumour types. The number of altered loci was used to develop a decision tree classifier model trained on the retrospectively collected cohort of 107 clinical cases sequenced by the CCCP assay.ResultsThe classifier was able to correctly classify all cases and was then used to analyse a test set of clinical cases (n=112) and was able to correctly predict their MSI status with 100% sensitivity and specificity. Analysis of recurrently altered loci identified alterations in genes involved in DNA repair, signalling and transcriptional regulation pathways, many of which have been implicated in MSI tumours.ConclusionThis study highlights the utility of this approach, which should be applicable to laboratories performing similar testing.

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Next-generation sequencing for tumor mutation quantification using liquid biopsies

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2019-0745 ◽

2020 ◽

Vol 58 (2) ◽

pp. 306-313 ◽

Cited By ~ 1

Author(s):

Mariano Provencio ◽

Clara Pérez-Barrios ◽

Miguel Barquin ◽

Virginia Calvo ◽

Fabio Franco ◽

...

Keyword(s):

Lung Cancer ◽

Next Generation Sequencing ◽

Correlation Coefficient ◽

Resistance Mutation ◽

Response To Treatment ◽

Clinical Samples ◽

Next Generation ◽

Liquid Biopsies ◽

Nsclc Patients ◽

Generation Sequencing

AbstractBackgroundNon-small cell lung cancer (NSCLC) patients benefit from targeted therapies both in first- and second-line treatment. Nevertheless, molecular profiling of lung cancer tumors after first disease progression is seldom performed. The analysis of circulating tumor DNA (ctDNA) enables not only non-invasive biomarker testing but also monitoring tumor response to treatment. Digital PCR (dPCR), although a robust approach, only enables the analysis of a limited number of mutations. Next-generation sequencing (NGS), on the other hand, enables the analysis of significantly greater numbers of mutations.MethodsA total of 54 circulating free DNA (cfDNA) samples from 52 NSCLC patients and two healthy donors were analyzed by NGS using the Oncomine™ Lung cfDNA Assay kit and dPCR.ResultsLin’s concordance correlation coefficient and Pearson’s correlation coefficient between mutant allele frequencies (MAFs) assessed by NGS and dPCR revealed a positive and linear relationship between the two data sets (ρc = 0.986; 95% confidence interval [CI] = 0.975–0.991; r = 0.987; p < 0.0001, respectively), indicating an excellent concordance between both measurements. Similarly, the agreement between NGS and dPCR for the detection of the resistance mutation p.T790M was almost perfect (K = 0.81; 95% CI = 0.62–0.99), with an excellent correlation in terms of MAFs (ρc = 0.991; 95% CI = 0.981–0.992 and Pearson’s r = 0.998; p < 0.0001). Importantly, cfDNA sequencing was successful using as low as 10 ng cfDNA input.ConclusionsMAFs assessed by NGS were highly correlated with MAFs assessed by dPCR, demonstrating that NGS is a robust technique for ctDNA quantification using clinical samples, thereby allowing for dynamic genomic surveillance in the era of precision medicine.

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