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 < 0.001). Seventy-five percent of the Ct-values were ≥ 33 in the egg-negative category, < 31 in the light intensity category, and < 24 in the heavy intensity category.

Novel tools and strategies for breaking schistosomiasis transmission: study protocol for an intervention study

“…Second, the urine filtration and reagent strip methods that we will apply for the standard diagnosis of S. haematobium infection markers are not very sensitively and specifically detecting light intensity infections as primarily found in Zanzibar [ 29 – 31 , 61 ]. Hence, in cross-sectional surveys, as well as in the surveillance-response approach, we will likely miss a considerable number of S. haematobium- positive individuals and underestimate the “true” prevalence.…”

“…Hence, 10 ml from each urine samples collected at the baseline and endline survey will be stored at -20 °C and examined with a highly sensitive and specific test at the end of the study. This might be a DNA-based PCR approach [ 31 ] or an antigen-based test such as the up-converting phosphor-lateral flow circulating anodic antigen (UCP-LF) CAA assay [ 30 ] or any other test with excellent parameters developed until 2024.…”

“…Moving towards schistosomiasis elimination, there is an enhanced need for sensitive and specific diagnostic tests that are high-throughput, and affordable and applicable at the point of care to assess reliably S. haematobium infections, prevalences and incidence [ 13 , 43 ]. An accurate picture of the endemic situation is important for programmatic decision making, to tailor specific intervention packages in line with (yet to be developed) target thresholds to those in need, to determine correctly the performance and impact of interventions in elimination settings, and to document sustained elimination [ 13 , 29 – 31 , 44 ].…”

Assessment of bacterial and fungal populations in urine from clinically healthy dogs using next‐generation sequencing

“…Clinically healthy dogs (n = 50) were prospectively identified by emailing a recruitment flyer to all faculty, staff, and students at West- Research Corp., Irvine, California) that preserves the microbial profile (microbiota) for prolonged periods at ambient temperature. 8 Consequently, all urine samples destined for microbiome analysis were batched and stored at 4 C until processing. Preserved samples were subsequently delivered to the MiDOG LLC testing facility (Irvine, California) and processed/sequenced as described in Section 2.2.…”

Bulinus senegalensis and Bulinus umbilicatus Snail Infestations by the Schistosoma haematobium Group in Niakhar, Senegal

“…The remaining negative samples from the RD-PCR could therefore be other species of the haematobium -group or hybrids strains. However, although the RD-PCR technique is very sensitive [ 28 ], we do not exclude the fact that it may not have amplified some samples positive for Dra 1 which is also known for its high sensitivity [ 35 ]. It should also be noted that the small number of samples tested is a limitation and does not further enhance our understanding of the RD-PCR test’s sensitivity.…”