Circulating cell-free DNA as a biomarker for diagnosis of Schistosomiasis japonica

Abstract

Background: Schistosomiasis, a neglected tropical disease, remains an important public health problem. Although there are various methods for diagnosing schistosomiasis, many limitations still exist. Early diagnosis and treatment of schistosomiasis can significantly improve survival and prognosis of patients.

Methodology: Circulating cell-free (cf)DNA has been widely used in the diagnosis of various diseases. In our study, we evaluated the diagnostic value of circulating cfDNA for schistosomiasis caused by Schistosoma japonicum. We focused on the tandem sequences and mitochondrial genes of S. japonicum to identify highly sensitive and specific targets for diagnosis of Schistosomiasis japonica.

Results: Through data screening and analysis, we ultimately identified four specific tandem sequences (TD-1, TD-2, TD-3. and TD-4) and six mitochondrial genes (COX1(1), COX1(2), CYTB, ATP6, COX3, and ND5). We designed specific primers to detect the amount of circulating cfDNA in S. japonicum-infected mouse and chronic schistosomiasis patients. Our results showed that the number of tandem sequences was significantly higher than that of the mitochondrial genes. A S. japonicum infection model in mice suggested that infection of S. japonicum can be diagnosed by detecting circulating cfDNA as early as the first week. We measured the expression levels of circulating cfDNA (TD-1, TD-2, and TD-3) at different time points and found that TD-3 expression was significantly higher than that of TD-1 or TD-2. We also infected mice with different quantities of cercariae (20 s and 80 s). The level of cfDNA (TD-3) in the 80 s infection group was significantly higher than in the 20 s infection group. Additionally, cfDNA (TD-3) levels increased after egg deposition. Meanwhile, we tested 42 patients with chronic Schistosomiasis japonica and circulating cfDNA (TD-3) was detected in nine patients.

Conclusions: We have screened highly sensitive targets for the diagnosis of Schistosomiasis japonica, and the detection of circulating cfDNA is a rapid and effective method for the diagnosis of Schistosomiasis japonica. The levels of cfDNA is correlated with cercariae infection severity. Early detection and diagnosis of schistosomiasis is crucial for patient treatment and improving prognosis.

Keywords: Schistosomiasis japonica; Diagnosis; cfDNA.

Fig. 1

The flowchart for screening candidate targets for diagnosis of Schistosomiasis japonicum. Tandem sequences and mitochondrial genes of Schistosoma japonicum were focused to identify highly sensitive and specific targets for Schistosomiasis japonica. Through data screening and analysis, we ultimately obtained four pairs of tandem primers and six pairs of mitochondrial gene primers

Fig. 2

PCR amplification of targets for diagnosis of Schistosomiasis japonicum. A we examined the expression of four tandem sequences (TD-1, TD-2, TD-3, and TD-4) and six mitochondrial genes (COX1(1), COX1(2), CYTB, ATP6, COX3, and ND5) in the liver of S. japonicum-infected mice at 8 weeks. B In both livers of normal mice (NC) and S. japonicum-infected mice (SJ) at 8 weeks, we detected the expression of TD-1, TD-2, and TD-3

Fig. 3

PCR amplification of targets in schistosome, lung flukes, and Toxoplasma gondii. To explore the specificity of TD-1, TD-2, and TD-3, we detect our screened targets (TD-1, TD-2, and TD-3) in other parasites, including lung flukes and Toxoplasma gondii.

Fig. 4

The sequence of Sanger sequencing about TD-1, TD-2, and TD-3. To verify the specificity of the TD-1, TD-2, and TD-3 sequences, we sequenced the amplified DNA and verified the TD-1, TD-2, and TD-3 sequences by Sanger sequencing

Fig. 5

Detection of cfDNA (TD-1, TD-2, and TD-3) in infected mice at different weeks. After extracting cfDNA from plasma of S. japonicum-infected mice at 1 week, 3 week, 5 week, and 8 week (n = 5), we detected the expression of TD-1, TD-2, and TD-3 (n = 5). The significance level was tested by unpaired t-test and the data are shown as the mean ± s.d. values (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 6

Detection of our targets and other reported targets. A The plot of PCR amplification of TD-1, TD-2, TD-3, SjR2, ITS2, and CYTB in the livers of S. japonicum-infected mice at 8 weeks. B The expression level comparison between TD-3 and other targets in the livers of S. japonicum-infected mice at 8 weeks (n = 5). The significance level was tested by unpaired t-test and the data are shown as the mean ± s.d. values (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Fig. 7

Detection of cfDNA (TD-3) in different number of cercariae infection. We infected mice with different quantities of cercariae (20 s and 80 s) and detected the levels of cfDNA (TD-3) at three weeks and eight weeks after cercariae infection(n = 5). The significance level was tested by unpaired t-test and the data are shown as the mean ± s.d. values (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)