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

Abstract

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 PLOS NEGLECTED TROPICAL DISEASES

Cell-free DNA as a diagnostic tool for human echinococcosis

“…Moradi et al concluded that their method of targeting long fragments could have missed the target as Echinococcus probably releases shorter fragments of cfDNA (90-200 bp) into the blood circulation [8]. Another study indeed found that the E. granulosus cfDNA in CE patients' plasma was mainly 100-350 bp, and that the E. multilocularis cfDNA in AE patients' plasma had a peak of 135 bp (50-330 bp), showing that the Echinococcus cfDNA in echinococcosis patients' blood occurs in shorter fragments [10].…”

“…However, this conclusion was based on one paper [6]. In recent years there have been several publications on the detection of Echinococcus cfDNA in the samples of echinococcosis patients (Table 1) [7][8][9][10][11][12][13].…”

“…As shown in Table 1 and Figure 1, there is Echinococcus cfDNA in echinococcosis patients' blood, although the concentration is very low [7][8][9][10]12,13]. The difference between the previous review and recent papers is mainly due to the methods used [5,6].…”

“…Weerakoon and McManus were citing the study of Chaya and Parija, which had targeted 450 bp of nad1, a classical mitochondrial gene [6]. However, other studies reported that most of the Echinococcus cfDNA in the blood of echinococcosis patients was from the nucleus, not from the mitochondrion [10,13]. Another possible reason for the discrepancy was that the target region of the NAAT might have been too long.…”

“…If there is agreement on the existence of Echinococcus cfDNA in echinococcosis patients' blood, the next question is: which sample type should be used? In the literature, both serum and plasma samples were used [6][7][8][9][10]12,13]. Plasma seemed to be a better sample for detecting Echinococcus cfDNA in echinococcosis patients' blood, with better sensitivities shown in more recent papers.…”