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.

Human infections by the gastrointestinal helminth Strongyloides stercoralis and the enteric protozoans Giardia duodenalis, Cryptosporidium spp. and Blastocystis spp. are not formally included in the list of 20 neglected tropical diseases prioritised by the World Health Organization. Although largely underdiagnosed and considered of lower public health relevance, these infections have been increasingly demonstrated to cause significant morbidity and even mortality globally, particularly among children living in resource-poor settings. In this cross-sectional survey the prevalence, frequency and molecular diversity of S. stercoralis, G. duodenalis, Cryptosporidium spp. and Blastocystis spp. were investigated in a school children population in the province of Benguela (Angola). A total of 351 stool samples were collected during January to June 2015. The presence of S. stercoralis and G. duodenalis was confirmed by qPCR methods. Giardia duodenalis assemblages and sub-assemblages were determined by multilocus sequence-based genotyping of the glutamate dehydrogenase and β-giardin genes of the parasite. Detection and identification of Cryptosporidium and Blastocystis species and subtypes was carried out by amplification and sequencing of a partial fragment of the small-subunit ribosomal RNA gene of both protozoan. Analyses of risk factors potentially associated with the transmission of these pathogens were also conducted. Prevalences of S. stercoralis, G. duodenalis, Cryptosporidium spp., and Blastocystis spp. were estimated at 21.4% (95% CI: 17.1–25.7%), 37.9% (95% CI: 32.8–43.0%), 2.9% (95% CI: 1.1–4.5%) and 25.6% (95% CI: 21.18–30.2%), respectively. Overall, 64.1% (225/351) of the children were infected by at least one of the pathogens investigated. Sequence analyses of the 28 G. duodenalis isolates that were successfully genotyped allowed the identification of sub-assemblages AI (14.3%), AII (14.3%), BIII (7.1%) and BIV (25.0%). Discordant typing results AII/AIII and BIII/BIV were identified in 7.1% and 14.3% of the isolates, respectively. A total of five additional isolates (17.9%) were identified as assemblage B. Three Cryptosporidium species including C. hominis (70%), C. parvum (20%) and C. canis (10%) were found circulating in the children population under study. A total of 75 Blastocystis isolates were assigned to the subtypes ST1 (30.7%), ST2 (30.7%), ST3 (36.0%), ST5 (1.3%) and ST7 (1.3%), respectively. Children younger than seven years of age had significantly higher risk of infections by protozoan enteropathogens (PRR: 1.35, P < 0.01), whereas being underweight seemed to have a protective effect against these infections (PRR: 0.74, P = 0.005). The burden of disease attributable to human strongyloidiasis, giardiosis, cryptosporidiosis and blastocystosis in Angola is considerably higher than initially estimated in previous surveys. Surveillance and control of these infections should be jointly tackled with formally considered neglected tropical diseases in order to maximize effort and available resources. Our data also demonstrate the added value of using molecular diagnostic methods in high transmission areas.

Schistosomiasis remains one of the most common endemic parasitic diseases affecting over 230 million people worlwide. Schistosoma mansoni is the main species causing intestinal and hepatic schistosomiasis and the fresh water pulmonate snails of the genus Biomphalaria are best known for their role as intermediate hosts of the parasite. The development of new molecular monitoring assays for large-scale screening of snails from transmission sites to detect the presence of schistosomes is an important point to consider for snail control interventions related to schistosomiasis elimination. Our work was focussed on developing and evaluating a new LAMP assay combined with a simple DNA extraction method to detect S. mansoni in experimentally infected snails as a diagnostic tool for field conditions. A LAMP assay using a set of six primers targeting a sequence of S. mansoni ribosomal intergenic spacer 28S-18S rRNA was designed. The detection limit of the LAMP assay was 0.1 fg of S. mansoni DNA at 63°C for 50 minutes. LAMP was evaluated by examining S. mansoni DNA in B. glabrata snails experimentally exposed to miracidia at different times post-exposure: early prepatent period (before cercarial shedding), light infections (snails exposed to a low number of miracidia) and detection of infected snails in pooled samples (within a group of uninfected snails). DNA for LAMP assays was obtained by using a commercial DNA extraction kit or a simple heat NaOH extraction method. We detected S. mansoni DNA in all groups of snails by using no complicated requirement procedure for DNA obtaining. Our LAMP assay, named Biompha-LAMP, is specific, sensitive, rapid and potentially adaptable as a cost-effective method for screening of intermediate hosts infected with S. mansoni in both individual snails and pooled samples. The assay could be suitable for large-scale field surveys for schistosomes control campaigns in endemic areas. Schistosoma mansoni is the main species causing intestinal and hepatic schistosomiasis worldwide and the snails of the genus Biomphalaria are best known for their role as intermediate hosts of the parasite. Molecular xenomonitoring for large-scale screening of snails from transmission sites to detect the presence of schistosomes is an important point to consider for snail control interventions related to schistosomiasis elimination. In our study, we have developed a new simple rapid LAMP assay to detect S. mansoni in Biomphalaria glabrata snails under different situations of infection: early prepatent period, light or low-grade infections and in snails pooled samples. Besides, a simple and rapid method for DNA extraction from snails' tissues was successfully used. This LAMP assay (named Biompha-LAMP) could be potentially useful for large-scale screening in searching infected snails with S. mansoni in field applicable conditions.

Strongyloides stercoralis, the chief causative agent of human strongyloidiasis, is a nematode globally distributed but mainly endemic in tropical and subtropical regions. Chronic infection is often clinically asymptomatic but it can result in severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. There is a great diversity of techniques used in diagnosing the disease, but definitive diagnosis is accomplished by parasitological examination of stool samples for morphological identification of parasite. Until now, no molecular method has been tested in urine samples as an alternative to stool samples for diagnosing strongyloidiasis. This study aimed to evaluate the use of a new molecular LAMP assay in a well-established Wistar rat experimental infection model using both stool and, for the first time, urine samples. The LAMP assay was also clinically evaluated in patients´ stool samples. Stool and urine samples were obtained daily during a 28-day period from rats infected subcutaneously with different infective third-stage larvae doses of S. venezuelensis. The dynamics of parasite infection was determined by daily counting the number of eggs per gram of feces from day 1 to 28 post-infection. A set of primers for LAMP assay based on a DNA partial sequence in the 18S rRNA gene from S. venezuelensis was designed. The set up LAMP assay (namely, Strong-LAMP) allowed the sensitive detection of S. venezuelensis DNA in both stool and urine samples obtained from each infection group of rats and was also effective in S. stercoralis DNA amplification in patients´ stool samples with previously confirmed strongyloidiasis by parasitological and real-time PCR tests. Our Strong-LAMP assay is an useful molecular tool in research of a strongyloidiasis experimental infection model in both stool and urine samples. After further validation, the Strong-LAMP could also be potentially applied for effective diagnosis of strongyloidiasis in a clinical setting. Human strongyloidiasis, a soil-transmitted infection mainly caused by Strongyloides stercoralis, is one of the most neglected among the so-called neglected tropical diseases (NTDs). The difficult diagnosis lead to an underreporting of infection rates. Strongyloidiasis can easily be misdiagnosed because many infections remain asymptomatic and the lack of sensitivity of the conventional fecal-based techniques for morphologically identification of infective larvae in feces. Although serologic tests are useful, a limitation in standardization to avoid cross-reactions still remains. There is an urgent need to improve more sensitive and specific diagnostic tests, particularly in immunocompromised patients or candidates to immunosuppressive treatments. Several molecular approaches for Strongyloides spp. DNA detection have already been assayed, but they have a very limited use in routine diagnostic, particularly in endemic areas. In addition, all molecular approaches for Strongyloides spp. DNA detection have always been mainly assayed for stool samples and no other more advantageous biological samples, such as urine, have been investigated for molecular purposes. In this study we have developed, for the first time, a molecular assay using LAMP methodology as a simple, sensible and robust method for the detection of S. venezuelensis DNA in a well-established Wistar rats experimental infection in both stool and urine samples. The LAMP assay was also successfully evaluated in patients´ stool samples. Our LAMP assay (Strong-LAMP) is an useful molecular tool in a strongyloidiasis experimental infection model and could be a potential field-friendly diagnostic test in a clinical setting, following further validation.