We preliminary assessed the DNA detection after bead beating using dilution of 2, 10, 50, and 90 eggs/10 mL of 3 urine samples with 131, 113, and 95 eggs/10 mL starting counts. For each sample of dilution, the extraction of DNA was performed by both procedures: Procedure A as conventional method without bead beating and Procedure B as new method with bead beating. The quality of DNA was tested by NanoVue Spectrophotometer (GE Healthcare) and good quality with A260/A280 ratio of 1.8–2.0 was obtained in all samples processed by both procedures A and B.
Then, each DNA specimen extracted from this preliminary assessment was analyzed by the Schistosoma spp. ITS2 Taqman real-time PCR (Obeng et al. 2008). As internal control for PCR inhibitors and amplification quality, the PhHV-1 analysis showed the expected Ct ≤ 32 and the amplification of each sample was considered not hampered by inhibitory factors. The Ct values were plotted against the starting eggs equivalent per reaction by performing linear regression analysis, and R2 of 0.92 and 0.82 was obtained from real-time PCR assay after extraction of DNA with and without bead beating, respectively (Fig. 1). Results showed the goodness of data fit of PCR assays for both procedures A and B, suggesting that the DNA detection becomes more effective independently from the PCR assay performance. Indeed, we calculated ∆Ct (Ct_Procedure A–Ct_Procedure B) per each point of egg concentration and we obtained ∆Ct 9, 8, 6, and 8 respectively for 2, 10, 50, and 90 eggs/10 mL, confirming the increase of DNA detection after bead beating.
Based on these results, we extended the comparison of DNA extraction with and without bead beating on 20 undiluted microscopy positive urine samples. We used 5 urine samples with no eggs detected at microscopy as true negative. For DNA extraction and real-time PCR, we followed identical conditions of procedures A and B as defined for the preliminary data described above. Among the 20 positive samples, we observed 5% high DNA load values (Ct < 25), 60% moderate (25 ≤ Ct ≤ 30), 20% low (30 < Ct < 40), and 15% not detected using the procedure A, while the procedure B showed 40% high, 40% moderate, and 20% low DNA load values, as shown in Fig. 2. Of note, only the procedure B provided 100% positivity. Table 1 reports the comparison between the median Ct values of the procedures with and without bead beating. The supplementary step of bead beating in procedure B showed a significant improved DNA detection in all samples analyzed. Moreover, the TaqMan assay resulted in specific detection of Schistosoma without any false-positive results, as shown by no DNA detection as expected on 5 negative urine samples. Similarly, negative controls (no DNA) provided no detection in each run (data not shown).
Schistosomiasis, mainly from S. mansoni and S. haematobium, is observed in non-endemic countries with increasing frequency. In Italy, the recent wave of asylum seekers from highly endemic areas has caused a “hidden epidemic” (Beltrame et al. 2017), with thousands of cases mostly undiagnosed, estimated to be currently living in the country. The sensitivity of direct microscopic examination as well as that of most antibody and antigen detection methods (such as CCA and CAA, mainly used for S. mansoni diagnosis) is unsatisfactory, as conventional PCR or real-time PCR, probably due to a lower average parasitic load if compared with endemic countries. The present findings suggest that a bead-beating procedure prior to DNA extraction has the potential to greatly increase S. haematobium DNA yield from urine. For the first assessment of this method, we used a limited sample size; a large-scale, prospective cohort study may provide more conclusive results from this method before indicating its routine use for the screening and diagnosis of imported urinary schistosomiasis. It would also be advisable that laboratories participate in an external quality assessment scheme, preferably a scheme using a proficiency panel of genuine clinical samples, to find out whether their DNA isolation procedure is sufficiently efficient.