Detection and genetic characterization of Echinococcus granulosus mitochondrial DNA in serum and formalin-fixed paraffin embedded cyst tissue samples of cystic echinococcosis patients

Abstract

Cystic echinococcosis (CE) is a worldwide zoonotic disease caused by the larval stage of Echinococcus granulosus. We investigated the presence of E. granulosus-specific DNA in the serum of CE patients by detecting the cytochrome c oxidase I (cox1) and NADH dehydrogenase subunit I (nad1) mitochondrial genes. Serum and formalin-fixed paraffin embedded (FFPE) cyst tissue samples of 80 CE patients were analyzed. The extracted DNA of samples was submitted to PCR amplification of cox1 and nad1 genes, and products were sequenced and genotyped. Nineteen (23.8%; 95% CI 15.8–34.1) serum and 78 (97.5%; 95% CI 91.3–99.3) FFPE cyst tissue samples were successfully amplified with at least one gene. Echinococcus DNA was detected in the sera of 15.0% (95% CI: 8.8–24.4) and 10.0% (95% CI: 5.2–18.5) and in cyst tissue of 91.3% (95% CI: 83.0–95.7) and 83.8% (95% CI: 74.2–90.3) of 80 patients by cox1 and nad1 gene, respectively. Four genotypes of E. granulosus were distinguished in the CE patients, with predominance of genotype G1, followed by G3, G2, and G6. The finding of E. granulosus DNA in 23.8% of serum samples from CE patients confirmed that E. granulosus releases cell-free DNA into the circulatory system, but quantities may be inadequate for the diagnosis of CE. Genotype G1 predominance suggests the sheep-dog cycle as the primary route of human infection.

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

“…DNA in the AE sample could be due to the mixture of necrotic parasite tissue and actively proliferating tissues. This is in line with the previous finding that the sensitivity of PCR-based methods in AE samples was higher than in CE samples [31][32][33]. As we only collected one AE sample, it needed further verification with more samples.…”

“…DNA in plasma is extremely low, whose proportion ranged 1.8e-5 to 4.0e-9 in these samples (Fig 2). Indeed, this low concentration may explain the low sensitivity of the PCR-based methods [31][32][33]. Besides the low concentration, we identified the difference between the cell-free Echinococcus spp.…”

“…Several attempts were made using cfDNA in Echinococcus spp. detection from plasma or serum with PCR-based methods, but their overall sensitivity was only 20% to 25% [31][32][33]. The low sensitivity could be due to non-existence, or low concentration of cfDNA of the parasite in the circulation, which showed our limited understanding of the cfDNA of Echinococcus spp.…”

“…The overall low proportion of mitochondrial-derived cell-free Echinococcus spp. DNA in plasma may also partially explain the low positive rate of mitochondrial gene based PCR [31][32][33]. However, reads per genome size of mitochondria were about 75.78 times larger than that of nuclear, which could be due to the multi copies of mitochondria [44].…”

“…Cell-free Echinococcus spp. DNA had already been suggested as a biomarker for echinococcosis [21], and its existence in plasma or serum was proven with PCR-based methods [31][32][33], though with rather low sensitivities (20-25%) [31][32][33] Low sensitivity prevents further application of using plasma cfDNA in the diagnosis of echinococcosis. The unsatisfactory performance of the previous attempts could be due to three possible reasons.…”