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 bio-marker 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 bio-marker 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 cellfree 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.