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

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. CONCLUSIONS: While the sensitivity of the qPCR was ~ 80% for very light intensity infections (egg counts < 10), in general, the Dra1 based qPCR assay detected twice as many S. haematobium infections compared with classical parasitological tests. The qPCR is hence a sensitive, urine-based approach for S. haematobium diagnosis that can be used for impact assessment of schistosomiasis elimination programmes, individual diagnosis, and in improved format also for verification and certification of elimination. TRIAL REGISTRATION: ISRCTN, ISRCTN48837681 . Registered 05 September 2012 - Retrospectively registered.

Development and validation of a multi-locus DNA metabarcoding method to identify endangered species in complex samples.

DNA metabarcoding provides great potential for species identification in complex samples such as food supplements and traditional medicines. Such a method would aid Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) enforcement officers to combat wildlife crime by preventing illegal trade of endangered plant and animal species. The objective of this research was to develop a multi-locus DNA metabarcoding method for forensic wildlife species identification and to evaluate the applicability and reproducibility of this approach across different laboratories. A DNA metabarcoding method was developed that makes use of 12 DNA barcode markers that have demonstrated universal applicability across a wide range of plant and animal taxa and that facilitate the identification of species in samples containing degraded DNA. The DNA metabarcoding method was developed based on Illumina MiSeq amplicon sequencing of well-defined experimental mixtures, for which a bioinformatics pipeline with user-friendly web-interface was developed. The performance of the DNA metabarcoding method was assessed in an international validation trial by 16 laboratories, in which the method was found to be highly reproducible and sensitive enough to identify species present in a mixture at 1% dry weight content. The advanced multi-locus DNA metabarcoding method assessed in this study provides reliable and detailed data on the composition of complex food products, including information on the presence of CITES-listed species. The method can provide improved resolution for species identification, while verifying species with multiple DNA barcodes contributes to an enhanced quality assurance.

Development of a multiplex DNA-based traceability tool for crop plant materials.

The authenticity of food is of increasing importance for producers, retailers and consumers. All groups benefit from the correct labelling of the contents of food products. Producers and retailers want to guarantee the origin of their products and check for adulteration with cheaper or inferior ingredients. Consumers are also more demanding about the origin of their food for various socioeconomic reasons. In contrast to this increasing demand, correct labelling has become much more complex because of global transportation networks of raw materials and processed food products. Within the European integrated research project 'Tracing the origin of food' (TRACE), a DNA-based multiplex detection tool was developed-the padlock probe ligation and microarray detection (PPLMD) tool. In this paper, this method is extended to a 15-plex traceability tool with a focus on products of commercial importance such as the emmer wheat Farro della Garfagnana (FdG) and Basmati rice. The specificity of 14 plant-related padlock probes was determined and initially validated in mixtures comprising seven or nine plant species/varieties. One nucleotide difference in target sequence was sufficient for the distinction between the presence or absence of a specific target. At least 5% FdG or Basmati rice was detected in mixtures with cheaper bread wheat or non-fragrant rice, respectively. The results suggested that even lower levels of (un-)intentional adulteration could be detected. PPLMD has been shown to be a useful tool for the detection of fraudulent/intentional admixtures in premium foods and is ready for the monitoring of correct labelling of premium foods worldwide.