A Blood Based Real-time PCR for Diagnosis of Schistosomiasis
A Blood Based Real-time PCR for Diagnosis of Schistosomiasis
Evaluation of blood based real-time PCR testing in travellers returning from schistosomiasis endemic regions with suspected acute schistosomiasis in 11 specialized European centres in a prospective study from January 2009 to April 2012. In detail the participating countries were Belgium, France, Norway and Germany. The sites are institutions specialised in tropical or travel medicine and are members of the EuroTravNet (http://www.istm.org/eurotravnet/main.html) initiated by The International Society of Travel Medicine and which represents a collaborative network of the European Centre of Disease Control (ECDC). The study was approved by the Hamburg Ethics Committee of the Chamber of Physicians and complied with the Declaration of Helsinki. For the Oslo site data managing files were slightly modified according to local data security requirements, for the other sites no changes were required.
Patients with a history of a recent travel to a schistosomiasis endemic region and freshwater contacts, an episode of fever (body temperature ≥ 38.5°C) and an absolute or relative eosinophil count of ≥700/μl or ≥10%, were eligible for participation (Group A; n = 38). In addition, 17 patients with high clinical suspect of acute schistosomiasis but without an episode of fever were also tested (Group B). An amount of at least 2 ml of serum was required for DNA extraction and PCR testing as well as for Schistosoma serology. Oral informed consent was obtained from every patient or their legal guardians.
Microscopy of faecal or urine samples for the detection of Schistosoma eggs were done on individual decision of the attending physician at each centre according to established in-house protocols. In general, stool samples were analysed using the formol-ether concentration technique. For detection of S. haematobium eggs, urine collected over a period of 24 hours or at least between 10 am and 2 pm was filtered and processed as described earlier.
Samples were sent to Hamburg by mail at room temperature and analysed for anti-Schistosoma specific antibodies and Schistosoma DNA.
All patient sera were tested for anti-schistosoma antibodies using an enzyme-immunoassay (EIA) and an immunofluorescence-assay (IFA), respectively.
DNA preparation from 2 ml serum was performed at the Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, using the QIAamp Circulating Nucleic Acid Kit according to the manufacturers suggestion (Qiagen, Hilden, Germany). Detection of cell-free Schistosoma DNA was performed according to a previously published protocol. The test targets the 121 bp tandem repeat sequence first described by Hamburger et al. using the primer sequences SRA1 (CCACGCTCTCGCAAATAATCT) and SRS2 (CAACCGTTCTATGAAAATCGTTGT) as previously reported. All PCRs were performed in duplicate. A test was considered positive when the threshold was attained within 45 PCR cycles (Ct-value < 45).
A patient was considered positive for acute schistosomiasis when either microscopy, serology or PCR testing revealed a positive result. Treatment with Praziquantel according to local protocols was offered to every patient with a positive test result.
Methods
Study Design
Evaluation of blood based real-time PCR testing in travellers returning from schistosomiasis endemic regions with suspected acute schistosomiasis in 11 specialized European centres in a prospective study from January 2009 to April 2012. In detail the participating countries were Belgium, France, Norway and Germany. The sites are institutions specialised in tropical or travel medicine and are members of the EuroTravNet (http://www.istm.org/eurotravnet/main.html) initiated by The International Society of Travel Medicine and which represents a collaborative network of the European Centre of Disease Control (ECDC). The study was approved by the Hamburg Ethics Committee of the Chamber of Physicians and complied with the Declaration of Helsinki. For the Oslo site data managing files were slightly modified according to local data security requirements, for the other sites no changes were required.
Inclusion Criteria
Patients with a history of a recent travel to a schistosomiasis endemic region and freshwater contacts, an episode of fever (body temperature ≥ 38.5°C) and an absolute or relative eosinophil count of ≥700/μl or ≥10%, were eligible for participation (Group A; n = 38). In addition, 17 patients with high clinical suspect of acute schistosomiasis but without an episode of fever were also tested (Group B). An amount of at least 2 ml of serum was required for DNA extraction and PCR testing as well as for Schistosoma serology. Oral informed consent was obtained from every patient or their legal guardians.
Microscopic Parasite Detection
Microscopy of faecal or urine samples for the detection of Schistosoma eggs were done on individual decision of the attending physician at each centre according to established in-house protocols. In general, stool samples were analysed using the formol-ether concentration technique. For detection of S. haematobium eggs, urine collected over a period of 24 hours or at least between 10 am and 2 pm was filtered and processed as described earlier.
Serum and Plasma Samples
Samples were sent to Hamburg by mail at room temperature and analysed for anti-Schistosoma specific antibodies and Schistosoma DNA.
Serology
All patient sera were tested for anti-schistosoma antibodies using an enzyme-immunoassay (EIA) and an immunofluorescence-assay (IFA), respectively.
DNA Preparation and Real Time PCR
DNA preparation from 2 ml serum was performed at the Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, using the QIAamp Circulating Nucleic Acid Kit according to the manufacturers suggestion (Qiagen, Hilden, Germany). Detection of cell-free Schistosoma DNA was performed according to a previously published protocol. The test targets the 121 bp tandem repeat sequence first described by Hamburger et al. using the primer sequences SRA1 (CCACGCTCTCGCAAATAATCT) and SRS2 (CAACCGTTCTATGAAAATCGTTGT) as previously reported. All PCRs were performed in duplicate. A test was considered positive when the threshold was attained within 45 PCR cycles (Ct-value < 45).
Diagnosis and Treatment
A patient was considered positive for acute schistosomiasis when either microscopy, serology or PCR testing revealed a positive result. Treatment with Praziquantel according to local protocols was offered to every patient with a positive test result.
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