Detection of Schistosoma mansoni DNA using polymerase chain reaction from serum and dried blood spot card samples of an adult population in North-western Tanzania

Background:Real-time polymerase chain reaction(PCR)is a sensitive and specific method for diagnosing schistosomiasis.However,this method should be performed in a laboratory,usually located distant from the sample collection site.Therefore,it is important to have fast sampling preservation methods,which allow simple transport prior to DNA extraction and amplification.The aim of this study was to verify if blood samples applied to filter paper are suitable for analysis of Schistosoma mansoni DNA by real-time PCR.Methods:A cross-sectional study was conducted among 100 study participants aged 17 to 70 years in a fishing village on the southern shore of Lake Victoria,fanzania.Serum samples and ethylenediaminetetraacetic acid(EDTA)-anticoagulated whole blood for preparation of dried blood spots(DBS)were collected to test for Schistosoma mansoni infection by real-time PCR.A combined diagnostic reference of positive results of serum-based real-time PCR and the Kato-Katz(KK)method was used for analysis.Sensitivity and negative predictive value(NPV)were calculated.The Wilcoxon signed-rank test was chosen to compare the mean cycle threshold(Ct)values from serum and DBS.Results:According to the reference,92.5%S.mansoni positive samples were determined.The serum-based real-time PCR performed excellently with 95.4%sensitivity,whereas the DBS-based real-time PCR showed a low sensitivity(45.4%).The Ct-values were significantly higher in DBS(median:37.3)than in serum samples(median:27.5,P<0.001),reflecting a lower parasite-specific DNA load on the filter cards.With increasing egg counts,an increase in sensitivity was observed for all methods.The POC-CCA test and the serum-based real-time PCR showed a sensitivity of 100%for medium and severe infections.The DBS real-time PCR showed a sensitivity of only 85.7%even for severe infections.Conclusions:DBS-based real-time PCR did not provide good results in our study and therefore should not be recommended or must be tested concerning temperature of storage,storage duration,use of different filter papers and extraction methods before it is used in future studies.In contrast,our results showed that the POC-CCA test is a sensitive and precise test for detecting S.mansoni infections.

Evaluation of serum-based real-time PCR to detect Schistosoma mansoni infection before and after treatment

Background:To detect acute schistosomiasis,low-intensity infections,or to verify the success of treatment with praziquantel,highly sensitive test methods are required.The aim of this study was therefore to demonstrate the performance of Schistosoma mansoni specific DNA detection in serum and urine using real-time polymerase chain reaction(PCR)in an endemic area before and after treatment.Methods:The study pursued a 1-week and 20-weeks longitudinal design with a treatment intervention among 36 study participants aged 18 to 70 years in the community of Kayenze,a fishing village in Ilemela district on the southern shore of Lake Victoria in north-western Tanzania between February and June 2018.Blood,urine and stool samples were collected from each participant to diagnose Schistosoma mansoni infection before and two times after treatment with praziquantel using serum-and urine based real-time PCR,point-of-care circulating cathodic antigen(POC-CCA)rapid diagnostic test and the microscopic Kato-Katz(KK)method.Kappa coefficient(κ)was used to estimate the agreement between these diagnostic tests compared to a combined“gold standard”of positive results by serum-based real-time PCR and/or positive egg counts determined by KK.Kendall’s Tau rank correlation was used to examine the relationship between cycle threshold(Ct)-values and egg counts and the Wilcoxon signed-rank test was used to compare the median Ct-values of the different examination time points.Results:By using the combined“gold standard”of the parasitological Kato-Katz test and/or serum-based real-time PCR,a S.mansoni prevalence of 77.1%could be determined at baseline.In terms of sensitivity,serum-based realtime PCR(96.3%)and POC-CCA assay(77.8%)showed the highest results.The detection of DNA from urine samples showed the lowest sensitivity(33.3%).Treatment with praziquantel resulted in a significantly reduced prevalence of S.mansoni.No infection could be detected by Kato-Katz,with the POC-CCA test only 33.3%.The analysis of the median Ct values over time(which were determined by the serum-based real-time PCR)showed that the Ct decreases significantly shortly after treatment(from 30.3 to 28)and increases above baseline level(34.9)three months later.Conclusions:The data presented here show that the serum-based real-time PCR exhibits excellent diagnostic accuracy,in contrast to the use of urine as sample material for S.mansoni DNA detection.However,as circulating DNA does not necessarily reflect the persistence of living worms in schistosomiasis,this method is less well suited to verify the success of treatment with praziquantel.