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Performance of a real-time PCR approach for diagnosing Schistosoma haematobium infections of different intensity in urine samples from Zanzibar.

ABSTRACT: BACKGROUND:Efforts to control and eliminate schistosomiasis have accelerated over the past decade. As parasite burden, associated morbidity and egg excretion decrease, diagnosis with standard parasitological methods becomes harder. We assessed the robustness and performance of a real-time PCR (qPCR) approach in comparison with urine filtration microscopy and reagent strip testing for the diagnosis of Schistosoma haematobium infections of different intensities. METHODS:The robustness of DNA isolation and qPCR was validated in eight laboratories from Europe and Africa. Subsequently, 792 urine samples collected during cross-sectional surveys of the Zanzibar Elimination of Schistosomiasis Transmission (ZEST) project in 2012-2017 were examined with qPCR in 2018. Diagnostic sensitivity of the qPCR was calculated at different infection intensity categories, using urine filtration microscopy as reference test. Spearman's rank correlation between Ct-values and S. haematobium egg counts was assessed and Ct-value percentiles for infection intensity categories determined. RESULTS:S. haematobium Dra1 DNA-positive samples were identified correctly in all eight laboratories. Examination of urine samples from Zanzibar revealed Dra1 DNA in 26.8% (212/792) by qPCR, S. haematobium eggs in 13.3% (105/792) by urine filtration, and microhaematuria in 13.8% (109/792) by reagent strips. Sensitivity of the qPCR increased with augmenting egg counts: 80.6% (29/36) for counts between 1 and 4 eggs, 83.3% (15/18) for counts between 5 and 9 eggs, 100% (23/23) for counts between 10 and 49 eggs, and 96.4% (27/28) for counts of 50+ eggs. There was a significant negative correlation between Ct-values and egg counts (Spearman's rho?=?-?0.49, P?

SUBMITTER: Keller D

PROVIDER: S-EPMC7487541 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Performance of a real-time PCR approach for diagnosing Schistosoma haematobium infections of different intensity in urine samples from Zanzibar.

Keller Dominique D Rothen Julian J Dangy Jean-Pierre JP Saner Corina C Daubenberger Claudia C Allan Fiona F Ame Shaali M SM Ali Said M SM Kabole Fatma F Hattendorf Jan J Rollinson David D Seyfarth Ralf R Knopp Stefanie S

Infectious diseases of poverty 20200904 1

<h4>Background</h4>Efforts to control and eliminate schistosomiasis have accelerated over the past decade. As parasite burden, associated morbidity and egg excretion decrease, diagnosis with standard parasitological methods becomes harder. We assessed the robustness and performance of a real-time PCR (qPCR) approach in comparison with urine filtration microscopy and reagent strip testing for the diagnosis of Schistosoma haematobium infections of different intensities.<h4>Methods</h4>The robustne ...[more]

PMID: 32887642

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Project description:BackgroundUrine filtration and microhaematuria reagent strips are basic standard diagnostic methods to detect urogenital schistosomiasis. We assessed their accuracy for the diagnosis of light intensity infections with Schistosoma haematobium as they occur in individuals living in Zanzibar, an area targeted for interruption of transmission.MethodsUrine samples were collected from children and adults in surveys conducted annually in Zanzibar from 2013 through 2016 and examined with the urine filtration method to count S. haematobium eggs and with the reagent strip test (Hemastix) to detect microhaematuria as a proxy for infection. Ten percent of the urine filtration slides were read twice. Sensitivity was calculated for reagent strips, stratified by egg counts reflecting light intensity sub-groups, and kappa statistics for the agreement of urine filtration readings.ResultsAmong the 39,207 and 18,155 urine samples examined from children and adults, respectively, 5.4% and 2.7% were S. haematobium egg-positive. A third (34.7%) and almost half (46.7%) of the egg-positive samples from children and adults, respectively, had ultra-low counts defined as 1-5 eggs per 10 ml urine. Sensitivity of the reagent strips increased significantly for each unit log10 egg count per 10 ml urine in children (odds ratio, OR: 4.7; 95% confidence interval, CI: 4.0-5.7; P < 0.0001) and adults (OR: 2.6; 95% CI: 1.9-3.7, P < 0.0001). Sensitivity for diagnosing ultra-light intensity infections was very low in children (50.1%; 95% CI: 46.5-53.8%) and adults (58.7%; 95% CI: 51.9-65.2%). Among the 4477 and 1566 urine filtration slides read twice from children and adults, most were correctly identified as negative or positive (kappa = 0.84 for children and kappa = 0.81 for adults). However, 294 and 75 slides had discrepant results and were positive in only one of the two readings. The majority of these discrepant slides (76.9% of children and 84.0% of adults) had counts of 1-5 eggs per 10 ml urine.ConclusionsWe found that many individuals infected with S. haematobium in Zanzibar excrete > 5 eggs per 10 ml urine. These ultra-light infections impose a major challenge for accurate diagnosis. Next-generation diagnostic tools to be used in settings where interruption of transmission is the goal should reliably detect infections with ? 5 eggs per 10 ml urine.Trial registrationISRCTN, ISRCTN48837681 . Registered 05 September 2012 - Retrospectively registered.

Project description:BackgroundIn an effort to enhance accuracy of diagnosis of Schistosoma haematobium, this study explores day-to-day variability and diagnostic performance of real-time PCR for detection and quantification of Schistosoma DNA compared to other diagnostic tools in an endemic area before and after treatment.MethodologyPreviously collected urine samples (N?=?390) from 114 preselected proven parasitological and/or clinical S. haematobium positive Kenyan schoolchildren were analyzed by a Schistosoma internal transcribed spacer-based real-time PCR after 14 years of storage. Pre-treatment day-to-day fluctuations of PCR and microscopy over three consecutive days were measured for 24 children using intra-class correlation coefficient. A combined 'gold standard' (PCR and/or microscopy positive) was used to measure sensitivity and negative predictive value (NPV) of several diagnostic tools at baseline, two and 18 months post-treatment with praziquantel.Principal findingsAll 24 repeatedly tested children were PCR-positive over three days with little daily variation in median Ct-values, while 83.3% were found to be egg-positive for S. haematobium at day 1 and 75.0% at day 2 and 3 pre-treatment, signifying daily fluctuations in microscopy diagnosis. Of all 114 preselected schoolchildren, repeated microscopic measurements were required to detect 96.5% versus 100% of positive pre-treatment cases by single PCR. At two months post-treatment, microscopy and PCR detected 22.8% versus 69.3% positive children, respectively. Based on the 'gold standard', PCR showed high sensitivity (>92%) as compared to >31% sensitivity for microscopy, both pre- and post-treatment.Conclusions/significanceDetection and quantification of Schistosoma DNA in urine by real-time PCR was shown to be a powerful and specific diagnostic tool for detection of S. haematobium infections, with less day-to-day variation and higher sensitivity compared to microscopy. The superior performance of PCR before, and two and 18 months post-treatment provides a compelling argument for PCR as an accurate and reproducible tool for monitoring treatment efficacy.

Dataset Information

Performance of a real-time PCR approach for diagnosing Schistosoma haematobium infections of different intensity in urine samples from Zanzibar.

Publications

Performance of a real-time PCR approach for diagnosing Schistosoma haematobium infections of different intensity in urine samples from Zanzibar.

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