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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. Author summary: Echinococcosis is one of the most neglected tropical diseases caused by the metacestodes of Echinococcus spp. tapeworms, which affect both humans and livestock. Plasma cell-free DNA (cfDNA) consists of nucleic acid fragments found extracellularly and may contain DNA released from the parasites. Research shows that a variety of parasites can be detected from plasma cfDNA. Cell-free Echinococcus spp. DNA in plasma or serum had been tested with PCR-based methods, but these PCR methods had low sensitivity ranged from 20% to 25%. Low sensitivity may be due to our limited understanding of cell-free Echinococcus spp. DNA in plasma. Here, we take advantage of high-throughput sequencing to get a comprehensive characterization of cell-free Echinococcus spp. DNA. Our results showed that with high-throughput sequencing we could detect cell-free Echinococcus spp. DNA in all samples, though at a very low level. Based on the sequencing data, we found that cell-free Echinococcus spp. DNA in plasma had a different fragment length distribution to cell-free human DNA, and fragment length distribution of cell-free Echinococcus spp. DNA is also different between cystic echinococcosis (CE) and alveolar echinococcosis (AE). The sequencing data can also help trace the release source of cell-free Echinococcus spp. DNA from the genome. According to the mapping results of cell-free Echinococcus spp. DNA reads, we found that most of them were from the nuclear genome rather than the mitochondrial genome, and their release position showed an even distribution on the genome. These characteristics of cell-free Echinococcus spp. DNA in echinococcosis patients' plasma could facilitate their future application in research or clinical settings. [ABSTRACT FROM AUTHOR]

Over one million people are living with cystic echinococcosis (CE) and alveolar echinococcosis (AE). For CE, long-term albendazole treatment is often needed, which requires regular follow-up. Follow-up is mainly through imaging which is insensitive to subtle changes and subjective to experience. We investigated the changes of Echinococcus granulosus (Eg) cell-free DNA (cfDNA) in plasma of CE patients before and after albendazole treatment to evaluate its potential as an objective marker for treatment follow-up. Plasma samples of nine CE patients were collected before and after treatment. We identified Eg cfDNA from every sample through high-throughput sequencing. Eg cfDNA concentration and fragment length increased significantly after the treatment period. Ultrasound examination before and after the treatment initiation reflected the drug effects to a certain extent, as the cyst size of four patients reduced. Our findings indicated that Eg cfDNA from plasma could be a potential marker in the monitoring of CE treatment. • Over 1,000,000 people are living with echinococcosis (mostly cystic echinococcosis). • For CE, long-term albendazole treatment is often needed with regular monitoring. • Monitoring through imaging is not objective and fails to identify subtle changes. • Echinococcus granulosus (Eg) cfDNA in CE patients' plasma changed after treatment. • Eg cfDNA in CE patients' plasma could be a potential marker in monitoring treatment. [ABSTRACT FROM AUTHOR]

RNA viruses are characterized by a broad host range and high levels of genetic diversity. Despite a recent expansion in the known virosphere following metagenomic sequencing, our knowledge of the species rank genetic diversity of RNA viruses, and how often they are misassigned and misclassified, is limited. We performed a clustering analysis of 7801 RNA-directed RNA polymerase (RdRp) sequences representing 1897 established RNA virus species. From this, we identified substantial genetic divergence within some virus species and inconsistency in RNA virus assignment between the GenBank database and The International Committee on Taxonomy of Viruses (ICTV). In particular, 27.57% virus species comprised multiple virus operational taxonomic units (vOTUs), including Alphainfluenzavirus influenzae, Mammarenavirus lassaense, Apple stem pitting virus , and Rotavirus A , with each having over 100 vOTUs. In addition, the distribution of average amino acid identity between vOTUs within single assigned species showed a relatively low threshold: <90% and sometimes <50%. However, when only exemplar sequences from virus species were analyzed, 1889 of the ICTV-designated RNA virus species (99.58%) were clustered into a single vOTU. Clustering of the RdRp sequences from different virus species also revealed that 17 vOTUs contained two distinct virus species. These potential misassignments were confirmed by phylogenetic analysis. A further analysis of average nucleotide identity (ANI) values ranging from 70% to 97.5% revealed that at an ANI of 82.5%, 1559 (82.18%) of the 1897 virus species could be correctly clustered into one single vOTU. However, at ANI values >82.5%, an increasing number of species were clustered into two or more vOTUs. In sum, we have identified some inconsistency and misassignment of the RNA virus species based on the analysis of RdRp sequences alone, which has important implications for the development of an automated RNA virus classification system., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press.)