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Background: Since its discovery in 1850 by Theodor Bilharz at Kasr Al-Ainy hospital, Cairo, Egypt, schistosomiasis continues to be a significant health challenge with a wide spectrum of chronic sequelae. There is no reliable single gold standard conventional method (microscopy or serology) for communities with low intensity and low transmission settings. Objectives: To evaluate a two-step immunomolecular diagnostic approach to rule-out recent Schistosoma re-infection in patients with characteristic pathological radiological features of chronic schistosomiasis. Subjects and Methods: Single serum samples were collected from patients from Kafr El Sheikh, Egypt. Selection of patients was based on microscopic absence of Schistosoma spp. eggs in either stool or urine samples; abstinence from treatment by Praziquantel in the last 2 months; and clinical and radiological diagnosis of pathological features of chronic schistosomiasis. All serum samples were serologically examined using indirect hemagglutination test (IHAT) for identification of anti-Schistosoma antibodies. Serum samples from 100 seropositive patients using IHAT were tested by multiplex PCR (mPCR) for detection of species-specific cell-free circulating Schistosoma DNA. Results: None of the seropositive serum samples were positive by PCR targeting Schistosoma speciesspecific gene. Patients who were farmers and/or who had received Praziquantel treatment were associated with Schistosoma seropositivity with statistical significance. Conclusion: A two-step immunomolecular diagnostic approach, using a single serum sample, can rule-out acute or active chronic schistosomiasis in patients that have characteristic pathological and radiological features of chronic schistosomiasis. Also, the two-step approach avoids over-treatment and development of drug resistance to Praziquantel, the only effective broad anti-Schistosoma medicine. Circulating Schistosoma DNA Gawish et al., 73 whether they are infected or not. In Egypt, since 1996, a widespread MDA schistosomiasis control program has been applied. This control program maintains low Schistosoma transmission and low intensity of infection, engendering the challenge of missed cases of acute schistosomiasis based on parasitological microscopy. In addition to misdiagnosis, microscopy is of limited value in diagnosis of schistosomiasis during chronic infections with a wide spectrum of chronic disease sequelae[3,9,10]. Diagnosing chronic schistosomiasis, in many countries including Egypt, is mainly dependent on serological diagnostic tests, mainly IHAT, in the presence of characteristic radiological image features and in the absence of detectable Schistosoma ova in urine, stool samples or tissue biopsy. Serological tests that depend upon antibody detection cannot differentiate between recent and old infection, persistent and re-infection, or co-occurrence of the two or more Schistosoma species. They are also limited by the variability in sensitivity and specificity of different serological test formats. In addition, antibodies persist for months or even years following clearance of infection[7,8]. Cell-free circulating Schistosoma-DNA are fragments detached from Schistosoma cells of a live-parasite and released in blood, saliva, urine, and other body fluids. Molecular detection of cell-free circulating SchistosomaDNA is used to detect early acute schistosomiasis and active chronic schistosomiasis in absence of Schistosoma eggs in stool and urine. These DNA fragments rapidly disappear within weeks following treatment making molecular methods a useful follow up tool[11-13]. This study’s intention was to evaluate a twostep immunomolecular diagnostic approach to rule-out Schistosoma recent re-infection in patients who had radiological characteristic pathological lesions of chronic schistosomiasis with a history of schistosomiasis or anti-Schistosoma treatment. SUBJECTS AND METHODS This laboratory-based cross-sectional study was carried out on serum samples from patients residing in Kafr El Sheikh from May, 2016 to February, 2017. Immunoand PCR assays were carried out in the Diagnostic and Research Unit of Parasitic Diseases (DRUP), the Molecular Medical Parasitology Lab (LMMP), respectively, Department of Medical Parasitology, Faculty of Medicine, Cairo University, Egypt. Patients: They were clinically and radiologically diagnosed with pathological features of chronic schistosomiasis and referred to the lab for serological testing for schistosomiasis. The exclusion criteria included patients less than 6 years old, patients with other underlying illnesses, patients for whom Schistosoma eggs were microscopically detected in their stool or urine samples and those who had taken Praziquantel in the last 2 months. A single blood sample was obtained from each patient, separated by centrifugation, then kept at -20oC for immunoassays and DNA extraction, followed by PCR assay. Related patients’ data were obtained with each sample and analyzed as predictors for Schistosoma seropositivity and molecular prevalence. Immunoassay: Part of the serum from all study individuals was examined for detection of antiSchistosoma antibodies using IHAT (Fumouze Diagnostics, France) following the manufacturer’s instructions. Molecular assay[14,15]: Serum samples from 100 seropositive patients using IHAT were tested by mPCR assay. DNA extraction of all serum samples was done using QIAamp DNA Blood Mini Kit (QiagenGermany) following instructions by the manufacturer. The mPCR was used to amplify the extracted genomicDNA targeting Schistosoma-species specific DNA mitochondrial cytochrome oxidase I (mtDNA) gene. It can differentiate between S. haematobium and S. mansoni based on length of mPCR products. The mPCR was performed following the reaction condition, reaction mix and a set of 3 primers[14,15], including common forward for Schistosoma (5`-TTT TTT GGT CAT CCT GAG GTG TAT-3), and 2 reverse primers for S. haematobium (5`-TGA TAA TCA ATG ACC CTG CAA TAA3′) and S. mansoni (5′-TGC AGA TAA AGC CAC CCC TGT G-3′). The mPCR products (543 bp for S. haematobium and 375 bp for S. mansoni) were electrophoresed on agarose gel (1.5%) after staining the gel by ethidium bromide and viewed by a UV transilluminator. Statistical analysis: Collected data was statistically analyzed using IBM SPSS V23 software. Patients associated quantitative variables were presented as mean and standard deviation; and the associated qualitative variables were tabulated as numbers and percentages. Variables were compared using the Chisquare test and/or Fisher exact test when appropriate. P value equal to or less than 0.5 was considered significant. Ethical Consideration: The study was started after approval by the ethical committee of the Faculty of Medicine, Al-Azhar University. Patients or their guardians were orally informed about the study goals; serum samples collection was done after acquiring their consent. Joining the study was voluntary.

parasitologists united journal

Circulating Schistosoma DNA in seropositive patients: A twostep diagnostic approach to rule-out acute and active chronic schistosomiasis in low transmission settings