16 Meanwhile this method has been adapted to suit smaller volumes (2 mL) of serum instead of 20 mL of plasma, without losing sensitivity. To perform a real-time PCR with sufficient specificity, a 121-bp tandem repeat sequence, identified by Hamburger in 1991, that consitutes about 12% of the S mansoni genome and is highly specific for all human schistosomes, was chosen as the real-time PCR target gene.17 Together with the patients’ test samples, a quantification standard plasmid (positive control), containing the nucleotides 39 to 79 bp from the 121-bp tandem repeat

sequence, and an internal negative control were run, as described by Panning.18 Plasmids were purified and subsequently TGF-beta inhibitor clinical trial quantified by spectrophotometry.

Dilutions of the standard plasmid were used as a quantification reference in real-time PCR. The cycle threshold value (Ct value) corresponds with the number of AG14699 PCR cycles needed to attain the threshold level of log-based fluorescence. A test was considered positive when the threshold was attained within 45 PCR cycles (Ct value <45). A lower Ct value corresponds with a higher amount of template DNA copies in the serum sample. In this cluster series, the specificity of the PCR assay for human schistosomes has not been assessed. This was already done by Wichmann in his original assay setup where he used plasma from 30 blood donors and 35 patients examined for other conditions to LY294002 be tested by large-volume plasma extraction and CFPD real-time PCR.16 None yielded positive results. We therefore felt it

unnecessary to have this experiment rerun. All patients were treated with a single dose of praziquantel at 40 mg/kg. Patients were instructed to take a single dose of methylpredisolone (0.5 mg/kg) in case fever developed soon after praziquantel treatment, and to contact the attending physician at our outpatient department for further advice regarding the duration of this treatment. Patients were seen again 5 weeks thereafter for evaluation of cure and a second single dose of praziquantel to be given to all, consistent with the established clinical practice at the outpatient clinic. For data analysis, a nonparametric test (Mann– Whitney/Wilcoxon) was used to compare continuous variables (including Ct values), and the Fisher exact test to compare proportions. Consistent with the in-house ethical guidelines for noninterventional studies, patients’ consent (or their adult guardians’ consent) was formally obtained to perform an additional diagnostic test on a preserved serum sample already used for antibody testing. The outcome of PCR test results did not interfere with the diagnosis, standard treatment, and follow-up procedure of schistosomiasis at the outpatient clinic.