Comprehensive characterization of plasma cell-free Echinococcus spp. DNA in echinococcosis patients using ultra-high-throughput sequencing

Background Echinococcosis is a life-threatening parasitic disease caused by Echinococcus spp. tapeworms with over one million people affected globally at any time. The Echinococcus spp. tapeworms in the human body release DNA to the circulatory system, which can be a biomarker for echinococcosis. Cell-free DNA (cfDNA) is widely used in medical research and has been applied in various clinical settings. As for echinococcosis, several PCR-based tests had been trialed to detect cell-free Echinococcus spp. DNA in plasma or serum, but the sensitivity was about 20% to 25%. Low sensitivity of PCR-based methods might be related to our limited understanding of the features of cell-free Echinococcus spp. DNA in plasma, including its concentration, fragment pattern and release source. In this study, we applied ultra-high-throughput sequencing to comprehensively investigate the characteristics of cell-free Echinococcus spp. DNA in plasma of echinococcosis patients. Methodology/Principal findings We collected plasma samples from 23 echinococcosis patients. Total plasma cfDNA was extracted and sequenced with a high-throughput sequencing platform. An average of 282 million read pairs were obtained for each plasma sample. Sequencing data were analyzed with bioinformatics workflow combined with Echinococcus spp. sequence database. After identification of cell-free Echinococcus spp. reads, we found that the cell-free Echinococcus spp. reads accounted for 1.8e-5 to 4.0e-9 of the total clean reads. Comparing fragment length distribution of cfDNA between Echinococcus spp. and humans showed that cell-free Echinococcus spp. DNA of cystic echinococcosis (CE) had a broad length range, while that of alveolar echinococcosis (AE) had an obvious peak at about 135 bp. We found that most of the cell-free Echinococcus spp. DNA reads were from the nuclear genome with an even distribution, which might indicate a random release pattern of cell-free Echinococcus spp. DNA. Conclusions/Significance With ultra-high-throughput sequencing technology, we analyzed the concentration, fragment length, release source, and other characteristics of cell-free Echinococcus spp. DNA in the plasma of echinococcosis patients. A better understanding of the characteristics of cell-free Echinococcus spp. DNA in plasma may facilitate their future application as a biomarker for diagnosis.

[1]  L. Akhlaghi,et al.  Detection and genetic characterization of Echinococcus granulosus mitochondrial DNA in serum and formalin-fixed paraffin embedded cyst tissue samples of cystic echinococcosis patients , 2019, PloS one.

[2]  M. Beer,et al.  Simple liver cysts and cystoid lesions in hepatic alveolar echinococcosis: a retrospective cohort study with Hounsfield analysis , 2019, Parasite.

[3]  Danielle M. Grant,et al.  European Echinococcus multilocularis Identified in Patients in Canada. , 2019, The New England journal of medicine.

[4]  Adriano Casulli,et al.  Echinococcus granulosus sensu lato. , 2019, Trends in parasitology.

[5]  Pasi K. Korhonen,et al.  Long-read sequencing reveals a 4.4 kb tandem repeat region in the mitogenome of Echinococcus granulosus (sensu stricto) genotype G1 , 2019, Parasites & Vectors.

[6]  Xiaonong Zhou,et al.  Epidemiological survey of echinococcosis in Tibet Autonomous Region of China , 2019, Infectious Diseases of Poverty.

[7]  D. McManus,et al.  Echinococcosis: Advances in the 21st Century , 2019, Clinical Microbiology Reviews.

[8]  Martin S. Lindner,et al.  Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease , 2019, Nature Microbiology.

[9]  Yi Feng,et al.  Performance of a Novel Low-Cost, Instrument-Free Plasma Separation Device for HIV Viral Load Quantification and Determination of Treatment Failure in People Living with HIV in Malaysia: a Diagnostic Accuracy Study , 2019, Journal of Clinical Microbiology.

[10]  Adriano Casulli,et al.  Echinococcus multilocularis. , 2019, Trends in parasitology.

[11]  C. Denkinger,et al.  Toward the Development of a Circulating Free DNA-Based In Vitro Diagnostic Test for Infectious Diseases: a Review of Evidence for Tuberculosis , 2018, Journal of Clinical Microbiology.

[12]  S. Joosse,et al.  Techniques of using circulating tumor DNA as a liquid biopsy component in cancer management , 2018, Computational and structural biotechnology journal.

[13]  Quanli Wang,et al.  meaRtools: An R package for the analysis of neuronal networks recorded on microelectrode arrays , 2018, bioRxiv.

[14]  T. Weber,et al.  Clinical management of cystic echinococcosis: state of the art and perspectives , 2018, Current opinion in infectious diseases.

[15]  C. Fìlice,et al.  Ultrasound and Cystic Echinococcosis , 2018, Ultrasound International Open.

[16]  Abel J Bronkhorst,et al.  The diverse origins of circulating cell‐free DNA in the human body: a critical re‐evaluation of the literature , 2018, Biological reviews of the Cambridge Philosophical Society.

[17]  Steven Salzberg,et al.  Removing contaminants from databases of draft genomes , 2018, PLoS Comput. Biol..

[18]  P. Peixoto,et al.  Circulating cell-free DNA in patients with alveolar echinococcosis. , 2018, Molecular and biochemical parasitology.

[19]  Jia Gu,et al.  fastp: an ultra-fast all-in-one FASTQ preprocessor , 2018, bioRxiv.

[20]  Sheng Chen,et al.  Efficient generation of complete sequences of MDR-encoding plasmids by rapid assembly of MinION barcoding sequencing data , 2018, GigaScience.

[21]  Jian Wang,et al.  SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data , 2017, GigaScience.

[22]  N. Nagarajan,et al.  Measurement of fetal fraction in cell-free DNA from maternal plasma using a panel of insertion/deletion polymorphisms , 2017, PloS one.

[23]  Richard A. Moore,et al.  Increasing quality, throughput and speed of sample preparation for strand-specific messenger RNA sequencing , 2017, BMC Genomics.

[24]  W. Kratzer,et al.  Imaging in alveolar echinococcosis (AE): Comparison of Echinococcus multilocularis classification for computed-tomography (EMUC-CT) and ultrasonography (EMUC-US) , 2017 .

[25]  M. Schaldenbrand,et al.  Multiorgan Echinococcus infection: Treatment of an immigrant in the United States , 2017, IDCases.

[26]  E. Brunetti,et al.  Is there echinococcosis in West Africa? A refugee from Niger with a liver cyst , 2017, Parasites & Vectors.

[27]  N. Rinehart,et al.  Physical activity, sedentary behavior and their correlates in children with Autism Spectrum Disorder: A systematic review , 2017, PloS one.

[28]  A. Salim,et al.  The Echinococcus canadensis (G7) genome: a key knowledge of parasitic platyhelminth human diseases , 2017, BMC Genomics.

[29]  C. Budke,et al.  The Echinococcoses: Diagnosis, Clinical Management and Burden of Disease. , 2017, Advances in parasitology.

[30]  A. Frary,et al.  Genome-wide SNP discovery and QTL mapping for fruit quality traits in inbred backcross lines (IBLs) of solanum pimpinellifolium using genotyping by sequencing , 2016, BMC Genomics.

[31]  U. Demir,et al.  Current status of diagnosis and treatment of hepatic echinococcosis , 2016, World journal of hepatology.

[32]  P. Jiang,et al.  The Long and Short of Circulating Cell-Free DNA and the Ins and Outs of Molecular Diagnostics. , 2016, Trends in genetics : TIG.

[33]  D. McManus,et al.  Cell-Free DNA as a Diagnostic Tool for Human Parasitic Infections. , 2016, Trends in parasitology.

[34]  E. Yılmaz,et al.  Efficacy of ultrasound-guided core-needle biopsy in the diagnosis of hepatic alveolar echinococcosis: a retrospective analysis , 2016, Parasite.

[35]  G. Baldi,et al.  Cystic echinococcosis in South America: systematic review of species and genotypes of Echinococcus granulosus sensu lato in humans and natural domestic hosts , 2016, Tropical medicine & international health : TM & IH.

[36]  C. Tinelli,et al.  Factors Influencing the Serological Response in Hepatic Echinococcus granulosus Infection. , 2016, The American journal of tropical medicine and hygiene.

[37]  E. Brunetti,et al.  Cystic Echinococcosis , 2015, Journal of Clinical Microbiology.

[38]  R. Manzano-Román,et al.  Serological Diagnosis and Follow-Up of Human Cystic Echinococcosis: A New Hope for the Future? , 2015, BioMed research international.

[39]  Sarah L. Kinnings,et al.  Factors affecting levels of circulating cell‐free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing , 2015, Prenatal diagnosis.

[40]  V. Wong,et al.  Lengthening and shortening of plasma DNA in hepatocellular carcinoma patients , 2015, Proceedings of the National Academy of Sciences.

[41]  Emanuela Locci,et al.  Monitoring the Modifications of the Vitreous Humor Metabolite Profile after Death: An Animal Model , 2015, BioMed research international.

[42]  Peter Ulz,et al.  Circulating tumor DNA as a liquid biopsy for cancer. , 2015, Clinical chemistry.

[43]  F. Grenouillet,et al.  Echinococcus metacestode: in search of viability markers , 2014, Parasite.

[44]  L. Carvalho,et al.  Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems , 2014, Memorias do Instituto Oswaldo Cruz.

[45]  L. Akhlaghi,et al.  Detection of Plasmodium vivax and Plasmodium falciparum DNA in human saliva and urine: loop-mediated isothermal amplification for malaria diagnosis. , 2014, Acta tropica.

[46]  Gary J. W. Liao,et al.  Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing , 2014, Proceedings of the National Academy of Sciences.

[47]  S. Parija,et al.  Performance of polymerase chain reaction for the diagnosis of cystic echinococcosis using serum, urine, and cyst fluid samples , 2014, Tropical parasitology.

[48]  Derrick E. Wood,et al.  Kraken: ultrafast metagenomic sequence classification using exact alignments , 2014, Genome Biology.

[49]  Shengyue Wang,et al.  The genome of the hydatid tapeworm Echinococcus granulosus , 2013, Nature Genetics.

[50]  A. Oliphant,et al.  Gestational age and maternal weight effects on fetal cell‐free DNA in maternal plasma , 2013, Prenatal diagnosis.

[51]  K. Shaheen,et al.  Erratum to “Small Intestinal Ischemia with Pneumatosis in a Young Adult: What Could Be the Cause?” , 2013, Case Reports in Gastrointestinal Medicine.

[52]  B. Boufana,et al.  Development of three PCR assays for the differentiation between Echinococcus shiquicus, E. granulosus (G1 genotype), and E. multilocularis DNA in the co-endemic region of Qinghai-Tibet plateau, China. , 2013, The American journal of tropical medicine and hygiene.

[53]  Jacqueline A. Keane,et al.  The genomes of four tapeworm species reveal adaptations to parasitism , 2013, Nature.

[54]  E. Brunetti,et al.  Extrahepatic Textiloma Long Misdiagnosed as Calcified Echinococcal Cyst , 2013, Case reports in gastrointestinal medicine.

[55]  B. Levy,et al.  Noninvasive prenatal testing: the future is now. , 2013, Reviews in obstetrics & gynecology.

[56]  N. Rosenfeld,et al.  Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing of Plasma DNA , 2012, Science Translational Medicine.

[57]  Richard M. Karp,et al.  Faster and More Accurate Sequence Alignment with SNAP , 2011, ArXiv.

[58]  D. Low,et al.  Polymerase chain reaction detection of Leishmania kDNA from the urine of Peruvian patients with cutaneous and mucocutaneous leishmaniasis. , 2011, The American journal of tropical medicine and hygiene.

[59]  Robert A. Edwards,et al.  Quality control and preprocessing of metagenomic datasets , 2011, Bioinform..

[60]  Yama W. L. Zheng,et al.  Maternal Plasma DNA Sequencing Reveals the Genome-Wide Genetic and Mutational Profile of the Fetus , 2010, Science Translational Medicine.

[61]  A. D. da Silva.  Human Echinococcosis: A Neglected Disease , 2010, Gastroenterology research and practice.

[62]  T. Billiar,et al.  Toll-Like Receptors in Hepatic Ischemia/Reperfusion and Transplantation , 2010, Gastroenterology research and practice.

[63]  E. Brunetti,et al.  Expert consensus for the diagnosis and treatment of cystic and alveolar echinococcosis in humans. , 2010, Acta tropica.

[64]  Aaron R. Quinlan,et al.  Bioinformatics Applications Note Genome Analysis Bedtools: a Flexible Suite of Utilities for Comparing Genomic Features , 2022 .

[65]  Ning Ma,et al.  BLAST+: architecture and applications , 2009, BMC Bioinformatics.

[66]  E. Brunetti,et al.  Update on cystic hydatid disease , 2009, Current opinion in infectious diseases.

[67]  Steven J. M. Jones,et al.  Circos: an information aesthetic for comparative genomics. , 2009, Genome research.

[68]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[69]  C. Grevelding,et al.  Diagnosing Schistosomiasis by Detection of Cell-Free Parasite DNA in Human Plasma , 2009, PLoS neglected tropical diseases.

[70]  P. J. Pretorius,et al.  The origin of circulating free DNA. , 2007, Clinical chemistry.

[71]  M. Spreeuwenberg,et al.  The Origin of Circulating Free DNA , 2007 .

[72]  D. McManus,et al.  Recent advances in the immunology and diagnosis of echinococcosis. , 2006, FEMS immunology and medical microbiology.

[73]  D. Carmena,et al.  Antigens for the immunodiagnosis of Echinococcus granulosus infection: An update. , 2006, Acta tropica.

[74]  S. Leung,et al.  Molecular characterization of circulating EBV DNA in the plasma of nasopharyngeal carcinoma and lymphoma patients. , 2003, Cancer research.

[75]  M. Kantarci,et al.  Hydatid disease from head to toe. , 2003, Radiographics : a review publication of the Radiological Society of North America, Inc.

[76]  J. Doležel,et al.  Nuclear DNA content and genome size of trout and human. , 2003, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[77]  J. Doležel,et al.  Nuclear DNA content and genome size of trout and human. Author's reply , 2003 .

[78]  G. Russomando,et al.  Polymerase chain reaction-based detection of Trypanosoma cruzi DNA in serum , 1992, Journal of clinical microbiology.