Human schistosomiasis, mainly due to Schistosoma mansoni species, is one of the most prevalent parasitic diseases worldwide. To overcome the drawbacks of classical parasitological and serological methods in detecting S. mansoni infections, especially in acute stage of the disease, development of cost-effective, simple and rapid molecular methods is still needed for the diagnosis of schistosomiasis. A promising approach is the loop-mediated isothermal amplification (LAMP) technology. Compared to PCR-based assays, LAMP has the advantages of reaction simplicity, rapidity, specificity, cost-effectiveness and higher amplification efficiency. Additionally, as results can be inspected by the naked eye, the technique has great potential for use in low-income countries. A sequence corresponding to a mitochondrial S. mansoni minisatellite DNA region was selected as a target for designing a LAMP-based method to detect S. mansoni DNA in stool samples. We used a S. mansoni murine model to obtain well defined stool and sera samples from infected mice with S. mansoni cercariae. Samples were taken weekly from week 0 to 8 post-infection and the Kato-Katz and ELISA techniques were used for monitoring the infection. Primer set designed were tested using a commercial reaction mixture for LAMP assay and an in house mixture to compare results. Specificity of LAMP was tested using 16 DNA samples from different parasites, including several Schistosoma species, and no cross-reactions were found. The detection limit of our LAMP assay (SmMIT-LAMP) was 1 fg of S. mansoni DNA. When testing stool samples from infected mice the SmMIT-LAMP detected S. mansoni DNA as soon as 1 week post-infection. We have developed, for the first time, a cost-effective, easy to perform, specific and sensitive LAMP assay for early detection of S. mansoni in stool samples. The method is potentially and readily adaptable for field diagnosis and disease surveillance in schistosomiasis-endemic areas. Schistosomiasis is one of the most widespread of all human parasitic diseases, Schistosoma mansoni being the most important species causing human intestinal schistosomiasis. The diagnosis of the disease is mainly based on parasitological and serological methods, but they are not effective in detecting S. mansoni infections in the acute stage of the disease. New diagnostic tools to detect the disease during the first weeks would be desirable, permitting early treatment and preventing the pathology associated with chronic infections. An approach is the loop-mediated isothermal amplification (LAMP) technique, which can amplify DNA with high specificity and sensitivity under isothermal conditions. DNA amplification and reading of results require minimum equipment, thus the technique has great potential for use in diagnosis of neglected tropical diseases. In our study, we developed and evaluated a LAMP assay for the early detection of S. mansoni DNA in stool samples from mice experimentally infected with the parasite. The results indicated that our LAMP assay is specific, sensitive and cost-effective in detecting S. mansoni DNA in stool samples as soon as one week post-infection, when parasitological and serological methods are not effective. The assay has the potential to be developed further as a field diagnostic tool for use in schistosomiasis-endemic areas.

Urogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis. A LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21% -85.63%) and 100% specificity (95% CI: 93.08%-100%). We have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid-Heat LAMPellet method and has the potential to be developed further as a field diagnostic tool for use in urogenital schistosomiasis-endemic areas. Human schistosomiasis is a disease caused by several species of parasitic worms of the genus Schistosoma that is affecting 200 million people, especially in sub-Saharan Africa. Most people are infected with Schistosoma haematobium, the species that causes urogenital schistosomiasis and also bladder cancer in many chronic infections. The definitive diagnostic test is based on microscopic examination of urine samples to detect parasite eggs. This method has low sensitivity, high day-to-day variability and cannot be carried out in the acute phase of the disease since the parasite has not started yet to lay eggs. New high-throughput diagnostic tools would be desirable, permitting early treatment and preventing the pathology associated with chronic infections. An interesting approach is the loop-mediated isothermal amplification (LAMP) technique because of its simplicity in operation and potential use in clinical diagnosis and surveillance of infectious diseases. In this study, we developed and evaluated a LAMP assay for detection of S. haematobium DNA in patients´ urine samples using heated pellets with no complicated requirement procedure for DNA extraction, namely the Rapid-Heat LAMPellet method. This is a new, easy, rapid and cost-effective LAMP method that should prove useful for mass screening in limited-resource settings in urogenital schistosomiasis-endemic areas.

In Brazil, schistosomiasis is a parasitic disease of public health relevance, mainly in poor areas where Schistosoma mansoni is the only human species encountered and Biomphalaria straminea is one of the intermediate host snails. A nested-PCR based on a specific mitochondrial S. mansoni minisatellite DNA region has been successfully developed and applied as a reference method in Brazil for S. mansoni detection, mainly in host snails for epidemiological studies. The amplification efficiency of LAMP is known to be higher than PCR. The present work aimed to assess the utility of our previously described SmMIT-LAMP assay for S. mansoni detection in human stool and snail samples in a low-transmission area of schistosomiasis in the municipality of Umbuzeiro, Paraíba State, Northeast Region of Brazil. A total of 427 human stool samples were collected during June-July 2016 in the municipality of Umbuzeiro and an overall prevalence of 3.04% (13/427) resulted positive by duplicate Kato-Katz thick smear. A total of 1,175 snails identified as Biomphalaria straminea were collected from 14 breeding sites along the Paraíba riverbank and distributed in 46 pools. DNA from human stool samples and pooled snails was extracted using the phenol/chloroform method. When performing the SmMIT-LAMP assay a total of 49/162 (30.24%) stool samples resulted positive, including 12/13 (92.31%) that were Kato-Katz positive and 37/149 (24.83%) previously Kato-Katz negative. By nested-PCR, only 1/46 pooled DNA snail samples was positive. By SmMIT-LAMP assay, the same sample also resulted positive and an additional one was positive from a different breeding site. Data of human and snail surveys were used to build risk maps of schistosomiasis incidence using kernel density analysis. This is the first study in which a LAMP assay was evaluated in both human stool and snail samples from a low-transmission schistosomiasis-endemic area. Our SmMIT-LAMP proved to be much more efficient in detection of S. mansoni in comparison to the 'gold standard' Kato-Katz method in human stool samples and the reference molecular nested-PCR in snails. The SmMIT-LAMP has demonstrated to be a useful molecular tool to identify potential foci of transmission in order to build risk maps of schistosomiasis. In Brazil, around 1.8 million people, mostly in the northeastern region of the country, are thought to be infected with Schistosoma mansoni. Snails of the genus Biomphalaria serve as intermediate hosts of the S. mansoni. A special program for schistosomiasis control was implemented more than 40 years ago in Brazil, decreasing prevalence, morbidity, and mortality over the past years. PCR-based diagnostic methods have been successfully applied in a few endemic areas of schistosomiasis in Brazil, although they are not still widely used due to the highly technical requirements making them unviable for routine application in field conditions. Loop-mediated isothermal amplification (LAMP) technology could be a powerful tool to apply for point-of-care testing in resource-poor settings. In previous work, a LAMP-based method to detect S. mansoni DNA, called SmMIT-LAMP, was developed by our research group to detect S. mansoni DNA testing stool samples from experimentally infected mice. Here, with the aim to apply SmMIT-LAMP as a cost-effective molecular tool for the detection of S. mansoni in field applicable conditions, we assess SmMIT-LAMP in human and snail samples collected in an endemic area of Brazil. The results obtained by Kato-Katz analysis of human stool samples and nested-PCR performed in snails were compared with the SmMIT-LAMP assay. It is the first time that a LAMP-based method has been used to identify potential transmission foci and to evaluate the epidemiological risk of acquiring schistosomiasis.

Schistosomiasis is one of the most prevalent Neglected Tropical Disease, affecting approximately 250 million people worldwide. Schistosoma mansoni is the most important species causing human intestinal schistosomiasis. Despite significant efforts in recent decades, the global disease burden of schistosomiasis remains extremely high. This could partly be attributed to the absence of accurate diagnostic tools, primarily in endemic areas. Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as a field-friendly alternative to many other complex molecular methods and it has been proposed as an ideal candidate for revolutionizing point-of-care molecular diagnostics. In a previous work, a LAMP-based method to detect S. mansoni DNA (SmMIT-LAMP) was developed by our research group for early diagnosis of active schistosomiasis in an experimental infection murine model. The SmMIT-LAMP has been further successfully evaluated in both human stool and snail samples and, recently, in human urine samples. In this study, we developed an important improvement for SmMIT-LAMP molecular assay, transforming it into a cold maintenance dry format suitable for potentially manufacturing as kit for ready-to-use for schistosomiasis diagnosis. This procedure could be applied to create dry LAMP kits for a laboratory setting and for diagnostic applications for other neglected tropical diseases.

The loop-mediated isothermal amplification (LAMP) technique is a great alternative to PCR-based methods, as it is fast, easy to use and works with high sensitivity and specificity without the need for expensive instruments. However, one of the limitations of LAMP is difficulty in achieving the simultaneous detection of several targets in a single tube, as the methodologies that allow this rely on fluorogenic probes containing specific target sequences, complicating their adaptation and the optimization of assays. Here, we summarize different methods for the development of multiplex LAMP assays based on sequence-specific detection, illustrated with a schematic representation of the technique, and evaluate their practical application based on the real-time detection and quantification of results, the possibility to visualize the results at a glance, the prior stabilization of reaction components, promoting the point-of-care use, the maximum number of specific targets amplified, and the validation of the technique in clinical samples. The various LAMP multiplexing methodologies differ in their operating conditions and mechanism. Each methodology has its advantages and disadvantages, and the choice among them will depend on specific application interests.