Diagnosis of Taenia solium infections based on “mail order” RNA-sequencing of single tapeworm egg isolates from stool samples v1

2021 ◽  
Author(s):  
Henrik Sadlowski ◽  
Veronika Schmidt ◽  
Jonathan Hiss ◽  
Christian G. Schneider ◽  
Gideon Zulu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Taenia Solium ◽  
Mail Order ◽  
Next Generation ◽  
Sample Collection ◽  
Additional Information ◽  
Stool Samples ◽  
Sequencing Facility ◽  
Generation Sequencing

Here we present a detailed protocol for the identification of Taenia solium based on the few Taenia spp. eggs found in diagnostic stool samples. Our approach is based on "mail order" RNA sequencing of single eggs and can be performed in laboratories equipped with basic tools such as a microscope, a Bunsen burner, and access to an international post office for shipping samples to a next-generation sequencing facility. This protocol describes sample collection and transport, isolation of individual Taenia spp. eggs, reliable disruption of individual Taenia eggs, and important considerations for shipping samples to a next-generation sequencing facility. We provide images and videos to help prepare the tools needed for the protocol. Additional information on our rationale for designing the critical steps can help implement the protocol in new environments.

Identification of Taenia solium based on single tapeworm eggs in diagnostic stool samples using RNA sequencing  v1

2020 ◽  
Author(s):  
Henrik Sadlowski ◽  
Veronika Schmidt ◽  
Jonathan Hiss ◽  
Christian G. Schneider ◽  
Gideon Zulu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Taenia Solium ◽  
Next Generation ◽  
Sample Collection ◽  
Additional Information ◽  
Stool Samples ◽  
Sequencing Facility ◽  
Generation Sequencing ◽  
Detailed Protocol

Here we present a detailed protocol for the identification of Taenia solium based on the few Taenia spp. eggs found in diagnostic stool samples. Our approach is based on "mail order" RNA sequencing of single eggs and can be performed in laboratories equipped with basic tools such as a microscope, a Bunsen burner, and access to an international post office for shipping samples to a next-generation sequencing facility. This protocol describes sample collection and transport, isolation of individual Taenia spp. eggs, reliable disruption of individual Taenia eggs, and important considerations for shipping samples to a next-generation sequencing facility. We provide images and videos to help prepare the tools needed for the protocol. Additional information on our rationale for designing the critical steps can help implement the protocol in new environments.

scholarly journals Diagnosis of Taenia solium infections based on “mail order” RNA-sequencing of single tapeworm egg isolates from stool samples

2021 ◽  
Vol 15 (12) ◽  
pp. e0009787
Author(s):  
Henrik Sadlowski ◽  
Veronika Schmidt ◽  
Jonathan Hiss ◽  
Johannes A. Kuehn ◽  
Christian G. Schneider ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Taenia Solium ◽  
Rna Extraction ◽  
Mitochondrial Rna ◽  
Mail Order ◽  
Laptop Computer ◽  
Diagnostic Laboratory ◽  
Community Health Programs ◽  
Stool Samples ◽  
Sequencing Facility

Combined community health programs aiming at health education, preventive anti-parasitic chemotherapy, and vaccination of pigs have proven their potential to regionally reduce and even eliminate Taenia solium infections that are associated with a high risk of neurological disease through ingestion of T. solium eggs. Yet it remains challenging to target T. solium endemic regions precisely or to make exact diagnoses in individual patients. One major reason is that the widely available stool microscopy may identify Taenia ssp. eggs in stool samples as such, but fails to distinguish between invasive (T. solium) and less invasive Taenia (T. saginata, T. asiatica, and T. hydatigena) species. The identification of Taenia ssp. eggs in routine stool samples often prompts a time-consuming and frequently unsuccessful epidemiologic workup in remote villages far away from a diagnostic laboratory. Here we present “mail order” single egg RNA-sequencing, a new method allowing the identification of the exact Taenia ssp. based on a few eggs found in routine diagnostic stool samples. We provide first T. solium transcriptome data, which show extremely high mitochondrial DNA (mtDNA) transcript counts that can be used for subspecies classification. “Mail order” RNA-sequencing can be administered by health personnel equipped with basic laboratory tools such as a microscope, a Bunsen burner, and access to an international post office for shipment of samples to a next generation sequencing facility. Our suggested workflow combines traditional stool microscopy, RNA-extraction from single Taenia eggs with mitochondrial RNA-sequencing, followed by bioinformatic processing with a basic laptop computer. The workflow could help to better target preventive healthcare measures and improve diagnostic specificity in individual patients based on incidental findings of Taenia ssp. eggs in diagnostic laboratories with limited resources.

scholarly journals Suitability of Bronchoscopic Biopsy Tissue Samples for Next-Generation Sequencing

Diagnostics ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 391
Author(s):  
Shuji Murakami ◽  
Tomoyuki Yokose ◽  
Daiji Nemoto ◽  
Masaki Suzuki ◽  
Ryou Usui ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Success Rate ◽  
Rna Sequencing ◽  
Surface Area ◽  
Endobronchial Ultrasound ◽  
Next Generation ◽  
Endobronchial Ultrasonography ◽  
Tissue Surface ◽  
The Impact ◽  
Generation Sequencing

A sufficiently large tissue sample is required to perform next-generation sequencing (NGS) with a high success rate, but the majority of patients with advanced non-small-cell lung cancer (NSCLC) are diagnosed with small biopsy specimens. Biopsy samples were collected from 184 patients with bronchoscopically diagnosed NSCLC. The tissue surface area, tumor cell count, and tumor content rate of each biopsy sample were evaluated. The impact of the cut-off criteria for the tissue surface area (≥1 mm2) and tumor content rate (≥30%) on the success rate of the Oncomine Dx Target Test (ODxTT) was evaluated. The mean tissue surface area of the transbronchial biopsies was 1.23 ± 0.85 mm2 when small endobronchial ultrasonography with a guide sheath (EBUS-GS) was used, 2.16 ± 1.49 mm2 with large EBUS-GS, and 1.81 ± 0.75 mm2 with endobronchial biopsy (EBB). The proportion of samples with a tissue surface area of ≥1 mm2 was 48.8% for small EBUS-GS, 79.2% for large EBUS-GS, and 78.6% for EBB. Sixty-nine patients underwent ODxTT. The success rate of DNA sequencing was 84.1% and that of RNA sequencing was 92.7% over all patients. The success rate of DNA (RNA) sequencing was 57.1% (71.4%) for small EBUS-GS (n = 14), 93.4% (96.9%) for large EBUS-GS (n = 32), 62.5% (100%) for EBB (n = 8), and 100% (100%) for endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) (n = 15). Regardless of the device used, a tissue surface area of ≥ 1 mm2 is adequate for samples to be tested with NGS.

scholarly journals Demystification of RNAseq Quality Control

2021 ◽  
Vol 22 (2) ◽  
Author(s):  
Dragana Dudić ◽  
Bojana Banović Đeri ◽  
Vesna Pajić ◽  
Gordana Pavlović-Lažetić
Keyword(s):  
Quality Control ◽  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Next Generation ◽  
Comprehensive Guidance ◽  
Dna And Rna ◽  
Control Evaluation ◽  
Downstream Analysis ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Next Generation Sequencing (NGS) analysis has become a widely used method for studying the structure of DNA and RNA, but complexity of the procedure leads to obtaining error-prone datasets which need to be cleansed in order to avoid misinterpretation of data. We address the usage and proper interpretations of characteristic metrics for RNA sequencing (RNAseq) quality control, implemented in and reported by FastQC, and provide a comprehensive guidance for their assessment in the context of total RNAseq quality control of Illumina raw reads. Additionally, we give recommendations how to adequately perform the quality control preprocessing step of raw total RNAseq Illumina reads according to the obtained results of the quality control evaluation step; the aim is to provide the best dataset to downstream analysis, rather than to get better FastQC results. We also tested effects of different preprocessing approaches to the downstream analysis and recommended the most suitable approach.

scholarly journals CBX2-dependent transcriptional landscape: implications for human sex development and its defects

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Patrick Sproll ◽  
Wassim Eid ◽  
Anna Biason-Lauber
Keyword(s):  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Current Knowledge ◽  
Next Generation ◽  
Steroidogenic Factor 1 ◽  
Sex Development ◽  
New Genes ◽  
Differences Of Sex Development ◽  
Generation Sequencing ◽  
Transcriptional Landscape

Abstract Sex development, a complex and indispensable process in all vertebrates, has still not been completely elucidated, although new genes involved in sex development are constantly being discovered and characterized. Chromobox Homolog 2 (CBX2) is one of these new additions and has been identified through a 46,XY girl with double heterozygous variants on CBX2.1, causing Differences of Sex Development (DSD). The mutated CBX2.1 failed to adequately regulate downstream targets important for sex development in humans, specifically steroidogenic factor 1 (NR5A1/SF1). To better place CBX2.1 in the human sex developmental cascade, we performed siRNA and CBX2.1 overexpression experiments and created a complete CRISPR/Cas9-CBX2 knockout in Sertoli-like cells. Furthermore, we deployed Next Generation Sequencing techniques, RNA-Sequencing and DamID-Sequencing, to identify new potential CBX2.1 downstream genes. The combination of these two next generation techniques enabled us to identify genes that are both bound and regulated by CBX2.1. This allowed us not only to expand our current knowledge about the influence of CBX2.1 in human sex development, but also to advance our insight in the mechanisms governing one of the most important decisions during embryonal development, the commitment to either female or male gonads.

Next-generation sequencing not superior to culture in periprosthetic joint infection diagnosis

2021 ◽  
Vol 103-B (1) ◽  
pp. 26-31
Author(s):  
Beau J. Kildow ◽  
Sean P. Ryan ◽  
Richard Danilkowicz ◽  
Alexander L. Lazarides ◽  
Colin Penrose ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Periprosthetic Joint Infection ◽  
Joint Infection ◽  
Next Generation ◽  
Sample Collection ◽  
Tissue Samples ◽  
Musculoskeletal Infection ◽  
Conventional Culture ◽  
Significant Difference ◽  
Generation Sequencing

Aims Use of molecular sequencing methods in periprosthetic joint infection (PJI) diagnosis and organism identification have gained popularity. Next-generation sequencing (NGS) is a potentially powerful tool that is now commercially available. The purpose of this study was to compare the diagnostic accuracy of NGS, polymerase chain reaction (PCR), conventional culture, the Musculoskeletal Infection Society (MSIS) criteria, and the recently proposed criteria by Parvizi et al in the diagnosis of PJI. Methods In this retrospective study, aspirates or tissue samples were collected in 30 revision and 86 primary arthroplasties for routine diagnostic investigation for PJI and sent to the laboratory for NGS and PCR. Concordance along with statistical differences between diagnostic studies were calculated. Results Using the MSIS criteria to diagnose PJI as the reference standard, the sensitivity and specificity of NGS were 60.9% and 89.9%, respectively, while culture resulted in sensitivity of 76.9% and specificity of 95.3%. PCR had a low sensitivity of 18.4%. There was no significant difference based on sample collection method (tissue swab or synovial fluid) (p = 0.760). There were 11 samples that were culture-positive and NGS-negative, of which eight met MSIS criteria for diagnosing infection. Conclusion In our series, NGS did not provide superior sensitivity or specificity results compared to culture. PCR has little utility as a standalone test for PJI diagnosis with a sensitivity of only 18.4%. Currently, several laboratory tests for PJI diagnosis should be obtained along with the overall clinical picture to help guide decision-making for PJI treatment. Cite this article: Bone Joint J 2021;103-B(1):26–31.

scholarly journals Next-generation sequencing combined with serological tests based pathogen analysis for a neurocysticercosis patient with a 20-year history:a case report

BMC Neurology ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Bin Chen ◽  
Zheng Chen ◽  
Yi-shu Yang ◽  
Gui-lan Cai ◽  
Xiao-jiao Xu ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Taenia Solium ◽  
Clinical Manifestations ◽  
Next Generation ◽  
Serum Samples ◽  
Igg Antibody ◽  
Serological Tests ◽  
Diagnostic Efficacy ◽  
Variable Sensitivity ◽  
Generation Sequencing

Abstract Background Neurocysticercosis (NCC) is the most common helminthic infection of the central nervous system (CNS) caused by the larval stage of Taenia solium. Accurate and early diagnosis of NCC remains challenging due to its heterogeneous clinical manifestations, neuroimaging deficits, variable sensitivity, and specificity of serological tests. Next-generation sequencing (NGS)-based pathogen analysis in patient’s cerebrospinal fluid (CSF) with NCC infection has recently been reported indicating its diagnostic efficacy. In this case study, we report the diagnosis of a NCC patient with a symptomatic history of over 20 years using NGS analysis and further confirmation of the pathology by immunological tests. Case presentation This study reports the clinical imaging and immunological features of a patient with a recurrent headache for more than 20 years, which worsened gradually with the symptom of fever for more than 7 years and paroxysmal amaurosis for more than 1 year. By utilizing NGS technique, the pathogen was detected in patient’s CSF, and the presence of Taenia solium-DNA was confirmed by a positive immunological reaction to cysticercus IgG antibody in CSF and serum samples. The symptoms of the patient were alleviated, and the CSF condition was improved substantially after the anti-helminthic treatment. Conclusions This study suggests that combining CSF NGS with cysticercus IgG testing may be a highly promising approach for diagnosing the challenging cases of NCC. Further studies are needed to evaluate the parasitic DNA load in patients’ CSF for the diagnosis of disease severity, stage, and monitoring of therapeutic responses.

scholarly journals Diagnosis and analysis of unexplained cases of childhood encephalitis in Australia using metagenomic next-generation sequencing

2021 ◽  
Author(s):  
Ci-Xiu Li ◽  
Rebecca Burrell ◽  
Russell C Dale ◽  
Alison Kesson ◽  
Christopher C Blyth ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Next Generation Sequencing ◽  
Rna Sequencing ◽  
Diagnostic Yield ◽  
Human Infection ◽  
Clinical Samples ◽  
Next Generation ◽  
Dna Viruses ◽  
Immune Mediated ◽  
Generation Sequencing

Encephalitis is most often caused by a variety of infectious agents, the identity of which is commonly determined through diagnostic tests utilising cerebrospinal fluid (CSF). Immune-mediated disorders are also a differential in encephalitis cases. We investigated the clinical characteristics and potential aetiological agents of unexplained encephalitis through metagenomic next-generation sequencing of residual clinical samples of multiple tissue types and independent clinical review. A total of 43 specimens, from both sterile and non-sterile sites, were collected from 18 encephalitis cases with no cause identified by the Australian Childhood Encephalitis study. Samples were subjected to total RNA sequencing to determine the presence and abundance of potential pathogens, to reveal mixed infections, pathogen genotypes, and epidemiological origins, and to describe the possible aetiologies of unexplained encephalitis. From this, we identified five RNA and two DNA viruses associated with human infection from both non-sterile (nasopharyngeal aspirates, nose/throat swabs, urine, stool rectal swab) and sterile (cerebrospinal fluid, blood) sites. These comprised two human rhinoviruses, two human seasonal coronaviruses, two polyomaviruses and one picobirnavirus. With the exception of picobirnavirus all have been previously associated with respiratory disease. Human rhinovirus and seasonal coronaviruses may be responsible for five of the encephalitis cases reported here. Immune-mediated encephalitis was considered clinically likely in six cases and RNA sequencing did not identify a possible pathogen in these cases. The aetiology remained unknown in nine cases. Our study emphasises the importance of respiratory viruses in the aetiology of unexplained child encephalitis and suggests that the routine inclusion of non-CNS sampling in encephalitis clinical guidelines/protocols could improve the diagnostic yield.

scholarly journals A statistical approach for tracking clonal dynamics in cancer using longitudinal next-generation sequencing data

2020 ◽  
Author(s):  
Dimitrios V. Vavoulis ◽  
Anthony Cutts ◽  
Jenny C. Taylor ◽  
Anna Schuh
Keyword(s):  
Next Generation Sequencing ◽  
Longitudinal Data ◽  
Darwinian Evolution ◽  
Next Generation Sequencing Data ◽  
Model Parameters ◽  
Next Generation ◽  
Sequencing Data ◽  
Sample Collection ◽  
Liquid Biopsies ◽  
Generation Sequencing

ABSTRACTTumours are composed of genotypically and phenotypically distinct cancer cell populations (clones), which are subject to a process of Darwinian evolution in response to changes in their local micro-environment, such as drug treatment. In a cancer patient, this process of continuous adaptation can be studied through next-generation sequencing of multiple tumour samples combined with appropriate bioinformatics and statistical methodologies. One family of statistical methods for clonal deconvolution seeks to identify groups of mutations and estimate the prevalence of each group in the tumour, while taking into account its purity and copy number profile. These methods have been used in the analysis of cross-sectional data, as well as for longitudinal data by discarding information on the timing of sample collection. Two key questions are how (in the case of longitudinal data) can we incorporate such information in our analyses and if there is any benefit in doing so. Regarding the first question, we incorporated information on the temporal spacing of longitudinally collected samples into standard non-parametric approaches for clonal deconvolution by modelling the time dependence of the prevalence of each clone as a Gaussian process. This permitted reconstruction of the temporal profile of the abundance of each clone continuously from several sparsely collected samples and without any strong prior assumptions on the functional form of this profile. Regarding the second question, we tested various model configurations on a range of whole genome, whole exome and targeted sequencing data from patients with chronic lymphocytic leukaemia, on liquid biopsy data from a patient with melanoma and on synthetic data. We demonstrate that incorporating temporal information in our analysis improves model performance, as long as data of sufficient volume and complexity are available for estimating free model parameters. We expect that our approach will be useful in cases where collecting a relatively long sequence of tumour samples is feasible, as in the case of liquid cancers (e.g. leukaemia) and liquid biopsies. The statistical methodology presented in this paper is freely available at github.com/dvav/clonosGP.

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