Search

Abstract Background Molecular markers are essential to identify Echinococcus species and genotypes in areas with multiple Echinococcus species to understand their epidemiology and pathology. Tibet Autonomous Region (TAR) is one of the areas worst hit by echinococcosis. However, molecular epidemiology is still missing among echinococcosis patients in TAR. This research explored the Echinococcus species and genotypes infecting humans in TAR and the population diversity and the possible origin of G1 in TAR. Methods Cyst samples were collected in one echinococcosis-designated hospital in TAR. Echinococcus species and genotypes were identified through a maximum-likelihood approach with near-complete/complete mtDNA using IQ-TREE. Phylogenetic networks were built with PopART, and the phylogeographical diffusion pattern was identified using a Bayesian discrete phylogeographic method. Results Using phylogenetic trees made with near-complete/complete mtDNA obtained from 92 cysts from TAR patients, the Echinococcus species and genotypes infecting humans in TAR were identified as Echinococcus granulosus (s.s.) G1 (81, 88.04%), accounting for the majority, followed by G6 of the E. canadensis cluster (6, 6.52%), E. granulosus (s.s.) G3 (3, 3.26%), and E. multilocularis (2, 2.17%). An expansion trend and a possible recent bottleneck event were confirmed among the G1 samples in TAR. Adding the other near-complete mtDNA of G1 samples globally from the literature, we identified the possible phylogeographic origin of the G1 samples in TAR as Turkey. Conclusions Using near-complete/complete mtDNA sequences of Echinococcus spp. obtained from echinococcosis patients, a variety of Echinococcus species and genotypes infecting humans throughout TAR were identified. As far as we know, this is the first comprehensive molecular investigation of Echinococcus species and genotypes infecting humans throughout TAR. We identified, for the first time to our knowledge, the possible origin of the G1 in TAR. We also enriched the long mtDNA database of Echinococcus spp. and added two complete E. multilocularis mtDNA sequences from human patients. These findings will improve our knowledge of echinococcosis, help to refine the targeted echinococcosis control measures, and serve as a valuable baseline for monitoring the Echinococcus species and genotypes mutations and trends of the Echinococcus spp. population in TAR. Graphical 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 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.

Introduction: Patients with clinically significant portal hypertension (CSPH) are recommended to be treated with non-selective beta-blockers (ie, carvedilol) to prevent the first hepatic decompensation event by the renewing Baveno VII consensus. CSPH is defined by hepatic venous pressure gradient (HVPG)≥10 mm Hg; however, the HVPG measurement is not widely adopted due to its invasiveness. Liver stiffness (LS)≥25 kPa can be used as a surrogate of HVPG≥10 mm Hg to rule in CSPH with 90% of the positive predicting value in majority aetiologies of patients. A compelling argument is existing for using LS≥25 kPa to diagnose CSPH and then to initiate carvedilol in patients with compensated cirrhosis, and about 5%-6% of patients under this diagnosis criteria may not be benefited from carvedilol and are at risk of lower heart rate and mean arterial pressure. Randomised controlled trial on the use of carvedilol to prevent liver decompensation in CSPH diagnosed by LS remains to elucidate. Therefore, we aimed to investigate if compensated cirrhosis patients with LS≥25 kPa may benefit from carvedilol therapy., Methods and Analysis: This study is a randomised, double-blind, placebo-controlled, multicentre trial. We will randomly assign 446 adult compensated cirrhosis patients with LS≥25 kPa and without any previous decompensated event and without high-risk gastro-oesophageal varices. Patients are randomly divided into two groups, with 223 subjects in group A and 223 subjects in group B. Group A is a carvedilol intervention group, while group B is a placebo group. All patients in both groups will receive aetiology therapies and are followed up at an interval of 6 months. The 3-year incidences of decompensated events of cirrhosis-related and liver-related death are the primary outcome. The secondary outcomes include development of each complication of portal hypertension individually (ascites, variceal bleeding or overt hepatic encephalopathy), development of spontaneous bacterial peritonitis and other bacterial infections, development of new varices, growth of small varices to large varices, delta changes in LS and spleen stiffness, change in hepatic dysfunction assessed by Child-Pugh and model for end-stage liver disease score, change in platelet count, development of hepatocellular carcinoma, development of portal vein thrombosis and adverse events with a 3-year follow-up. A predefined interim analysis will be performed to ensure that the calculation is reasonable., Ethics and Dissemination: The study protocol has been approved by the ethics committees of the Sixth People's Hospital of Shenyang (2023-05-003-01) and independent ethics committee for clinical research of Zhongda Hospital, affiliated to Southeast University (2023ZDSYLL433-P01). The results from this trial will be submitted for publication in peer-reviewed journals and will be presented at international conferences., Trial Registration Number: ChiCTR2300073864., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)