Diagnosing schistosomiasis by detection of cell-free parasite DNA in human plasma

Abstract

Introduction: Schistosomiasis (bilharzia), one of the most relevant parasitoses of humans, is confirmed by microscopic detection of eggs in stool, urine, or organ biopsies. The sensitivity of these procedures is variable due to fluctuation of egg shedding. Serological tests on the other hand do not distinguish between active and past disease. In patients with acute disease (Katayama syndrome), both serology and direct detection may produce false negative results. To overcome these obstacles, we developed a novel diagnostic strategy, following the rationale that Schistosoma DNA may be liberated as a result of parasite turnover and reach the blood. Cell-free parasite DNA (CFPD) was detected in plasma by PCR.

Methodology/principal findings: Real-time PCR with internal control was developed and optimized for detection of CFPD in human plasma. Distribution was studied in a mouse model for Schistosoma replication and elimination, as well as in human patients seen before and after treatment. CFPD was detectable in mouse plasma, and its concentration correlated with the course of anti-Schistosoma treatment. Humans with chronic disease and eggs in stool or urine (n = 14) showed a 100% rate of CFPD detection. CFPD was also detected in all (n = 8) patients with Katayama syndrome. Patients in whom no viable eggs could be detected and who had been treated for schistomiasis in the past (n = 30) showed lower detection rates (33.3%) and significantly lower CFPD concentrations. The duration from treatment to total elimination of CFPD from plasma was projected to exceed one year.

Conclusions/significance: PCR for detection of CFPD in human plasma may provide a new laboratory tool for diagnosing schistosomiasis in all phases of clinical disease, including the capacity to rule out Katayama syndrome and active disease. Further studies are needed to confirm the clinical usefulness of CFPD quantification in therapy monitoring.

Figure 1. DNA copies per mL of pooled mouse plasma (y-axis, four mice per datum point) in mice infected intraperitoneally with 100 cercariae of S. mansoni.

After completion of parasite maturation on day 42, mice were treated orally with praziquantel on day 45 (120 mg per kg). At the indicated times (x-axis), four mice were sacrificed, their blood pooled, and 1 mL of pooled plasma was tested as described in the Materials and Methods section for cell-free Schistosoma DNA. The untreated group is marked with an asterisk (*).

Figure 2. Cell-free Schistosoma DNA concentrations in plasma of patients with acute disease (Katayama syndrome) plotted against the days post exposure or post onset of symptoms when the tested samples were taken.

10 mL of plasma were tested for cell-free DNA.

Figure 3. Cell-free Schistosoma DNA concentrations after treatment.

DNA concentrations were plotted only for those patients still showing cell-free Schistosoma DNA in plasma after treatment. These data were pooled from patients who had been followed prospectively after being diagnosed with Katayama syndrome, as well as from patients examined retrospectively after concluded treatment. Linear regression analysis yielded the graph equation Y = 2.03−0.02 X. Exponential regression yielded the graph equation Y = e ^∧ −0.02 (X−30.4).

Figure 4. Box plot analysis of cell-free DNA concentrations in patients with Katayama syndrome (first visits only), patients with chronic disease, and all patients who had positive plasma PCR after treatment (pooled from treated Katayama syndrome patients and patients examined retrospectively after treatment).

Boxes represent the innermost two quartiles (25%–75% percentiles = interquartile range, IQR) of data. The whiskers represent an extension of the 25^th or 75^th percentiles by 1.5 times the IQR. The notches represent the median +/−1.57 IQR √n. If the notches of two boxes do not overlap, the medians ( = notch centers) are significantly different (true for the active disease vs. the treated group).