Echinococcus spp. and genotypes infecting humans in Tibet Autonomous Region of China: a molecular investigation with near-complete/complete mitochondrial sequences.

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.

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.

[Quality control method of Tibetan medicine Saussureae obvalltae].

To control the quality of Tibetan medicine Saussureae obvalltae, the quality control method and standard were established in this study. The water content, total ash, acid-insoluble ash and ethanol-soluble extractives of Saussureae obvalltae were determined according to the methods recorded in appendix of Chinese Pharmacopeia(2010 edition). Microscopical identification was performed for the plant and powder of the leaves. The TLC method was established, with chlorogenic acid and rutin as control substances, and a mixture of acetate-btuanone-formic acid-water (10∶6∶1∶2) as developing solvent and silica gel G as thin layer plate. Agilent ZORBAX Eclipse XDB-C18 (4.6 mm×250 mm, 5 µm) column was adopted and eluted with the mobile phase of acetonitrile-0.1% phosphoric acid (17∶83) in a gradient mode at a flow rate of 1.0 mL•min⁻¹. The column temperature was 40 ℃ and the detection wavelength was 350 nm. As a result, the plant and leaves of Saussureae obvalltae of different origins showed constent microscopic features. Chlorogenic acid, rutin and the other constituents were well separated on TLC detected under the sun light. According to the results of the methodological study, chlorogenic acid and rutin were in good linear correlation in the ranges of 0.119 2-0.715 4 µg(r=0.999 9) and 0.160 7-0.964 4 µg(r=1.000), and the average recoveries were 105.4% (RSD 1.4%) and 99.50% (RSD 0. 91%), respectively. The content of ethanol-soluble extractives, water content, total ash and acid-insoluble ash were 26.01%-31.59%, 7.16%-8.04%, 8.46%-11.14%,0.50%-1.87%, respectively. According to the study, the established method was specific and accurate, which could be used for the quality control of this drug.