Laboratory and field validation of the recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) for snail xenomonitoring for schistosomiasis.

Improvements in diagnostics for schistosomiasis in both humans and snail hosts are priorities to be able to reach the World Health Organization (WHO) goal of eliminating the disease as a public health problem by 2030. In this context, molecular isothermal amplification tests, such as Recombinase Polymerase Amplification (RPA), are promising for use in endemic areas at the point-of-need for their accuracy, robustness, simplicity, and time-effectiveness. The developed recombinase polymerase amplification assay targeting the Schistosoma mansoni mitochondrial minisatellite region (SmMIT-RPA) was used to detect S. mansoni DNA from both laboratory and field Biomphalaria snails. Laboratory snails were experimentally infected and used at one, seven, and 28 days post-exposure (dpe) to 10 S. mansoni miracidia to provide samples in the early pre-patent infection stage. Field samples of Biomphalaria spp. were collected from the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil, which are endemic for S. mansoni. The sensitivity and specificity of the SmMIT-RPA assay were analysed and compared with existing loop-mediated isothermal amplification (LAMP), PCR-based methods, parasitological examination of the snails, and nucleotide sequencing. The SmMIT-RPA assay was able to detect S. mansoni DNA in the experimentally infected Biomphalaria glabrata as early as one dpe to 10 miracidia. It also detected S. mansoni infections (55.5% prevalence) in the field samples with the highest accuracy (100% sensitivity and specificity) compared with the other molecular tests used as the reference. Results from this study indicate that the SmMIT-RPA assay is a good alternative test to be used for snail xenomonitoring of S. mansoni due to its high sensitivity, accuracy, and the possibility of detecting early pre-patent infection. Its simplicity and portability also make it a suitable methodology in low-resource settings.

Corrigendum: Assessment of the accuracy of 11 different diagnostic tests for the detection of Schistosomiasis mansoni in individuals from a Brazilian area of low endemicity using latent class analysis.

[This corrects the article DOI: 10.3389/fmicb.2022.1048457.].

Development of real-time and lateral flow recombinase polymerase amplification assays for rapid detection of Schistosoma mansoni.

Background: Accurate diagnosis followed by timely treatment is an effective strategy for the prevention of complications together with reducing schistosomiasis transmission. Recombinase Polymerase Amplification (RPA) is a simple, rapid, sensitive, and specific isothermal method with low resource needs. This research aimed at the development and optimisation of a real-time (RT) and a lateral flow (LF) RPA assay for the detection of Schistosoma mansoni. Methodology: Recombinase Polymerase Amplification reactions were performed at full- (as recommended) and half-volumes (to reduce costs), with RT or LF detection systems targeting the S. mansoni mitochondrial minisatellite region. The specificity was assessed using gDNA from other Schistosoma species, helminths co-endemic with S. mansoni, human stool, and urine, and Biomphalaria snail hosts. The analytical sensitivity was evaluated using serial dilutions of gDNA, synthetic copies of the target, and single eggs. The ability of both assays to detect the S. mansoni DNA in human urine and stool samples was also tested. The long-term stability of the RT-RPA reagents was evaluated by storing the reaction components in different temperature conditions for up to 3 weeks. Results: The RT- and the LF-RPA (SmMIT- and SmMIT-LF-RPA, respectively) presented similar results when used full- and half-volumes, thus the latter was followed in all experiments. The SmMIT-RPA was 100% specific to S. mansoni, able to detect a single egg, with a limit of detection (LOD) of down to 1 fg of gDNA and one synthetic copy of the target. The assay was able to detect S. mansoni DNA from stool containing 1 egg/g and in spiked urine at a concentration of 10 fg/µl. SmMIT-RPA reagents were stable for up to 3 weeks when kept at 19°C, and 2 weeks when stored at 27°C. The SmMIT-LF-RPA cross-reacted with Clinostomidae, presented the LOD of 10 fg and one synthetic copy of the target, being able to detect a single egg and 1 egg/g in a stool sample. The LOD in spiked urine samples was 10 pg/µl. Conclusion: The half-volume SmMIT-RPA is a promising method to be used in the field. It is specific, sensitive, robust, and tolerant to inhibitors, with a long-term stability of the reaction components and the real-time visualisation of results.

Assessment of the accuracy of 11 different diagnostic tests for the detection of Schistosomiasis mansoni in individuals from a Brazilian area of low endemicity using latent class analysis.

Background: Schistosomiasis is a parasitic disease associated with poverty. It is estimated that 7.1 million people are infected with Schistosoma mansoni in Latin America, with 95% of them living in Brazil. Accurate diagnosis and timely treatment are important measures to control and eliminate schistosomiasis, but diagnostic improvements are needed to detect infections, especially in areas of low endemicity. Methodology: This research aimed to evaluate the performance of 11 diagnostic tests using latent class analysis (LCA). A cross-sectional survey was undertaken in a low endemicity area of the municipality of Malacacheta, Minas Gerais, Brazil. Feces, urine, and blood samples were collected from 400 residents older than 6 years of age, who had not been treated with praziquantel in the 12 months previous to the collection of their samples. The collected samples were examined using parasitological (Helm Test® kit Kato-Katz), nucleic acid amplification tests -NAATs (PCR, qPCR and LAMP on urine; PCR-ELISA, qPCR and LAMP on stool), and immunological (POC-CCA, the commercial anti-Schistosoma mansoni IgG ELISA kit from Euroimmun, and two in-house ELISA assays using either the recombinant antigen PPE or the synthetic peptide Smp150390.1) tests. Results: The positivity rate of the 11 tests evaluated ranged from 5% (qPCR on urine) to 40.8% (commercial ELISA kit). The estimated prevalence of schistosomiasis was 12% (95% CI: 9-15%) according to the LCA. Among all tests assessed, the commercial ELISA kit had the highest estimated sensitivity (100%), while the Kato-Katz had the highest estimated specificity (99%). Based on the accuracy measures observed, we proposed three 2-step diagnostic approaches for the active search of infected people in endemic settings. The approaches proposed consist of combinations of commercial ELISA kit and NAATs tests performed on stool. All the approaches had higher sensitivity and specificity than the mean values observed for the 11 tests (70.4 and 89.5%, respectively). Conclusion: We showed that it is possible to achieve high specificity and sensitivity rates with lower costs by combining serological and NAATs tests, which would assist in the decision-making process for appropriate allocation of public funding aiming to achieve the WHO target of eliminating schistosomiasis as a public health problem by 2030.

A loop-mediated isothermal amplification assay for Schistosoma mansoni detection in Biomphalaria spp. from schistosomiasis-endemic areas in Minas Gerais, Brazil.

BACKGROUND: Schistosomiasis a neglected tropical disease  endemic in Brazil. It is caused by the trematode Schistosoma mansoni, which is transmitted by snails of the genus Biomphalaria. Among measures used to control and eliminate schistosomiasis, accurate mapping and monitoring of snail breeding sites are recommended. Despite the limitations of parasitological methods, they are still used to identify infected snails. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cost-effective diagnostic method for the identification of infected snails. In the work reported here, we aimed to validate the use of LAMP for the detection of S. mansoni in snails of the genus Biomphalaria. METHODS: Snails were collected in five municipalities of the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil. Snails were pooled according to collection site and then squeezed for the detection of S. mansoni and other trematode larvae. Pooled snails were subjected to pepsin digestion and DNA extraction. Molecular assays were performed for species-specific identification and characterization of the samples. A previously described LAMP assay was adapted, evaluated, and validated using laboratory and field samples. RESULTS: Using the parasitological method described here, S. mansoni cercariae were detected in snails from two collection sites, and cercariae of the family Spirorchiidae were found in snails from one site. The snails were identified by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites. Biomphalaria kuhniana, which is resistant to S. mansoni infection, was found in the remaining sites. Multiplex, low stringency (LS), and conventional PCR allowed the detection of positive snails in four additional sites. Trematodes belonging to the families Strigeidae and Echinostomatidae were detected by multiplex PCR in two sites. The LAMP assay was effective in detecting the presence of S. mansoni infection in laboratory (7 days post-infection) and field samples with no cross-reactivity for other trematodes. When compared to LS and conventional PCR, LAMP showed 100% specificity, 85.7% sensitivity, and a κ index of 0.88. CONCLUSIONS: Our findings suggest that LAMP is a good alternative method for the detection and monitoring of transmission foci of S. mansoni, as it was three times as effective as the parasitological examination used here for the detection of infection, and is more directly applicable in the field than other molecular techniques.

A multiplex PCR protocol for rapid differential identification of four families of trematodes with medical and veterinary importance transmitted by Biomphalaria Preston, 1910 snails.

Trematodes have complex life cycles with multiple hosts. Biomphalaria snails commonly act as the first intermediate hosts of several species that can affect human and animal health. The specific identification of larval trematodes found in snails is difficult and limited, since the taxonomy of these flukes is based on morphological traits of the adults found in vertebrates. Despite recent advances worldwide, studies aiming at the use of molecular tools for the identification of cercariae found in snails are scarce in the South America. In fact, most studies are focused on Schistosoma mansoni, with few efforts directed towards the identification of larvae of other parasites found in planorbids. When reported, these other parasites are identified as cercarial types, an artificial morphological system of classification. Therefore, alternative strategies for a correct, rapid and inexpensive identification of larval trematodes found in Biomphalaria are needed. This work aimed at developing a methodology capable of distinguishing four important families of trematodes (Clinostomidae, Echinostomatidae, Schistosomatidae and Strigeidae) commonly found infecting species of Biomphalaria. Using the rDNA sequences of 34 species as input for the online tool TipMT, we designed trematode family-specific primers targeting the ITS region optimized to be used in multiplex PCR. The panel of primers identified in this study was effective at the same PCR condition. The specificity of the primers was confirmed, and the PCR sensitivity ranged from 0.1 ng to 1 ag of the DNA of the parasite. This methodology was also effective for the detection of coinfection. Through a simple, fast, accurate, and inexpensive methodology, it is possible to properly identify the trematode families included in this study in a single PCR reaction. A family level identification provides important information about probable hosts, pattern of life cycle and possible impacts that the infection generates in a specific region, thus allowing the design of better control strategies, especially for those infections that have medical and veterinary importance.

Geographical distribution of intermediate hosts of Schistosoma mansoni in the states of Paraná, Minas Gerais, Bahia, Pernambuco and Rio Grande do Norte, Brazil, 2012-2014. / Distribuição geográfica dos hospedeiros intermediários do Schistosoma mansoni nos estados do Paraná, Minas Gerais, Bahia, Pernambuco e Rio Grande do Norte, 2012-2014.

OBJECTIVE: to describe the geographical distribution of intermediate hosts of Schistosoma mansoni in five Brazilian states. METHODS: this was a descriptive cross-sectional study; municipalities were selected in the states of Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) , and Rio Grande do Norte (98), for the period 2012 to 2014; these municipalities were chosen because they did not have current records of the presence of snails vectores de S. mansoni. The molluscs were captured and taxonomically identified and examined for S. mansoni cercariae. RESULTS: the work was carried out in 427 municipalities (99.5% of the 429 selected); the presence of mollusks was registered in 300 (70.2%) municipalities; Biomphalaria glabrata were found in 62 (21%) municipalities, B. straminea in 181 (60%), B. tenagophila in three (1%); B. glabrata/B. straminea association was found in 53 municipalities (18%) and B. glabrata/B. tenagophila association in one (0.3%) municipality. CONCLUSION: B. glabrata, B. straminea and B. tenagophila distribution records obtained in this study are consistent with previously known distribution.

Distribuição geográfica dos hospedeiros intermediários do Schistosoma mansoni nos estados do Paraná, Minas Gerais, Bahia, Pernambuco e Rio Grande do Norte, 2012-2014 / Distribución geográfica de los huéspedes intermedios de Schistosoma mansoni en los estados de Paraná, Minas Gerais, Bahia, Pernambuco y Rio Grande do Norte, Brasil, 2012-2014 / Geographical distribution of intermediate hosts of Schistosoma mansoni in the states of Paraná, Minas Gerais, Bahia, Pernambuco and Rio Grande do Norte, Brazil, 2012-2014

Objetivo: descrever a distribuição geográfica dos hospedeiros intermediários do Schistosoma mansoni em cinco estados brasileiros. Métodos: estudo transversal; foram selecionados municípios dos estados do Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) e Rio Grande do Norte (98), nos anos de 2012 a 2014; esses municípios foram escolhidos por não possuírem registros atualizados da presença de caramujos hospedeiros intermediários de S. mansoni; moluscos foram capturados, taxonomicamente identificados e examinados para verificação de cercárias de S. mansoni. Resultados: os trabalhos foram realizados em 427 municípios (99,5% dos 429 selecionados); foi registrada presença de moluscos em 300 (70,2%) municípios e a ocorrência de Biomphalaria glabrata em 62 (21%) municípios, B. straminea em 181 (60%), B. tenagophila em três (1%); associação de B. glabrata/B. straminea foi encontrada em 53 (18%), e de B. glabrata/B. tenagophila em um (0,3%). Conclusão: os registros de B. glabrata, B. straminea e B. tenagophila estão de acordo com a distribuição conhecida.

Objetivo: describir la distribución geográfica de los hospedadores intermediarios de Schistosoma mansoni en cinco estados brasileños. Métodos: estudio epidemiológico transversal; el estudio fue realizado en municipios de los estados de Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) y Rio Grande do Norte (98), entre 2012 y 2014; estos municipios fueron elegidos por no poseer registros actualizados de la presencia de caracoles vectores de S. mansoni; los moluscos fueron capturados, taxonómicamente identificados y examinados para la verificación de cercarias de S. mansoni. Resultados: los trabajos fueron realizados en 427 municipios (99,5% de 429 municipios seleccionados); fue registrada presencia de moluscos en 300 (70,2%) municípios; la presencia de Biomphalaria glabrata fue registrada en 62 (21%) municipios, B. straminea en 181 (60%) y B. tenagophila en três (1%); se observó asociación de B. glabrata con B. straminea en 53 (18%) y de B. glabrata con B. tenagophila en uno (0,3%). Conclusión: los registros de Biomphalaria están de acuerdo con la distribución conocida.

Objective: to describe the geographical distribution of intermediate hosts of Schistosoma mansoni in five Brazilian states. Methods: this was a descriptive cross-sectional study; municipalities were selected in the states of Paraná (78), Minas Gerais (120), Bahia (82), Pernambuco (51) , and Rio Grande do Norte (98), for the period 2012 to 2014; these municipalities were chosen because they did not have current records of the presence of snails vectores de S. mansoni. The molluscs were captured and taxonomically identified and examined for S. mansoni cercariae. Results: the work was carried out in 427 municipalities (99.5% of the 429 selected); the presence of mollusks was registered in 300 (70.2%) municipalities; Biomphalaria glabrata were found in 62 (21%) municipalities, B. straminea in 181 (60%), B. tenagophila in three (1%); B. glabrata/B. straminea association was found in 53 municipalities (18%) and B. glabrata/B. tenagophila association in one (0.3%) municipality. Conclusion: B. glabrata, B. straminea and B. tenagophila distribution records obtained in this study are consistent with previously known distribution.

Evaluation and updating of the Medical Malacology Collection (Fiocruz-CMM) using molecular taxonomy.

BACKGROUND: The Medical Malacology Collection (Coleção de Malacologia Médica, Fiocruz-CMM) is a depository of medically relevant mollusks, especially from the genus Biomphalaria, which includes the hosts of Schistosoma mansoni. Taxonomic studies of these snails have traditionally focused on the morphology of the reproductive system. However, determination of some species is complicated by the similarity shown by these characters. Molecular techniques have been used to try to overcome this problem. DESCRIPTION: The Fiocruz-CMM utilizes morphological and/or molecular method for species' identification. However, part of the collection has not been identified by molecular techniques and some points were unidentified. The present study employs polymerase chain reaction-based analysis of restriction fragment length polymorphisms (PCR-RFLP) to evaluate the identification of Biomphalaria in the Fiocruz-CMM, correct existing errors, assess the suitability of taxonomic synonyms, and identify unknown specimens. The results indicated that 56.7% of the mollusk specimens were correctly identified, 4.0% were wrongly identified, and 0.4% was identified under taxonomic synonyms. Additionally, the PCR-RFLP analysis identified for the first time 17.6% of the specimens in the Collection. However, 3.1% of the specimens could not be identified because the mollusk tissues were degraded, and 18.2% of the specimens were inconclusively identified, demonstrating the need for new taxonomic studies in this group. CONCLUSION: The data was utilized to update data of Environmental Information Reference Center (CRIA). These studies demonstrate the importance of using more than one technique in taxonomic confirmation and the good preservation of specimens' collection.