RESUMEN
Urogenital schistosomiasis is caused by the blood fluke Schistosoma haematobium and is one of the most neglected tropical diseases worldwide, afflicting > 100 million people. It is characterised by granulomata, fibrosis and calcification in urogenital tissues, and can lead to increased susceptibility to HIV/AIDS and squamous cell carcinoma of the bladder. To complement available treatment programs and break the transmission of disease, sound knowledge and understanding of the biology and ecology of S. haematobium is required. Hybridisation/introgression events and molecular variation among members of the S. haematobium-group might effect important biological and/or disease traits as well as the morbidity of disease and the effectiveness of control programs including mass drug administration. Here we report the first chromosome-contiguous genome for a well-defined laboratory line of this blood fluke. An exploration of this genome using transcriptomic data for all key developmental stages allowed us to refine gene models (including non-coding elements) and annotations, discover 'new' genes and transcription profiles for these stages, likely linked to development and/or pathogenesis. Molecular variation within S. haematobium among some geographical locations in Africa revealed unique genomic 'signatures' that matched species other than S. haematobium, indicating the occurrence of introgression events. The present reference genome (designated Shae.V3) and the findings from this study solidly underpin future functional genomic and molecular investigations of S. haematobium and accelerate systematic, large-scale population genomics investigations, with a focus on improved and sustained control of urogenital schistosomiasis.
Asunto(s)
Variación Genética , Genoma de Protozoos , Schistosoma haematobium/genética , Esquistosomiasis Urinaria/parasitología , Transcriptoma , Animales , Cromosomas/parasitología , Genes Protozoarios , Genoma , Estudio de Asociación del Genoma Completo , Análisis de Secuencia de ADNRESUMEN
Hybridization is a fascinating evolutionary phenomenon that raises the question of how species maintain their integrity. Inter-species hybridization occurs between certain Schistosoma species that can cause important public health and veterinary issues. In particular hybrids between Schistosoma haematobium and S. bovis associated with humans and animals respectively are frequently identified in Africa. Recent genomic evidence indicates that some S. haematobium populations show signatures of genomic introgression from S. bovis. Here, we conducted a genomic comparative study and investigated the genomic relationships between S. haematobium, S. bovis and their hybrids using 19 isolates originating from a wide geographical range over Africa, including samples initially classified as S. haematobium (n = 11), S. bovis (n = 6) and S. haematobium x S. bovis hybrids (n = 2). Based on a whole genomic sequencing approach, we developed 56,181 SNPs that allowed a clear differentiation of S. bovis isolates from a genomic cluster including all S. haematobium isolates and a natural S. haematobium-bovis hybrid. All the isolates from the S. haematobium cluster except the isolate from Madagascar harbored signatures of genomic introgression from S. bovis. Isolates from Corsica, Mali and Egypt harbored the S. bovis-like Invadolysin gene, an introgressed tract that has been previously detected in some introgressed S. haematobium populations from Niger. Together our results highlight the fact that introgression from S. bovis is widespread across S. haematobium and that the observed introgression is unidirectional.
Asunto(s)
Genoma , Hibridación Genética , Polimorfismo de Nucleótido Simple , Schistosoma haematobium/genética , Schistosoma/genética , Esquistosomiasis/parasitología , África , Animales , Caenorhabditis elegans , Schistosoma/clasificación , Schistosoma/aislamiento & purificación , Schistosoma haematobium/aislamiento & purificación , Esquistosomiasis/genética , Esquistosomiasis/patología , Especificidad de la Especie , Secuenciación Completa del GenomaRESUMEN
Schistosoma mansoni, a snail-borne, blood fluke that infects humans, was introduced into the Americas from Africa during the Trans-Atlantic slave trade. As this parasite shows strong specificity to the snail intermediate host, we expected that adaptation to South American Biomphalaria spp. snails would result in population bottlenecks and strong signatures of selection. We scored 475,081 single nucleotide variants in 143 S. mansoni from the Americas (Brazil, Guadeloupe and Puerto Rico) and Africa (Cameroon, Niger, Senegal, Tanzania, and Uganda), and used these data to ask: (i) Was there a population bottleneck during colonization? (ii) Can we identify signatures of selection associated with colonization? (iii) What were the source populations for colonizing parasites? We found a 2.4- to 2.9-fold reduction in diversity and much slower decay in linkage disequilibrium (LD) in parasites from East to West Africa. However, we observed similar nuclear diversity and LD in West Africa and Brazil, suggesting no strong bottlenecks and limited barriers to colonization. We identified five genome regions showing selection in the Americas, compared with three in West Africa and none in East Africa, which we speculate may reflect adaptation during colonization. Finally, we infer that unsampled populations from central African regions between Benin and Angola, with contributions from Niger, are probably the major source(s) for Brazilian S. mansoni. The absence of a bottleneck suggests that this is a rare case of a serendipitous invasion, where S. mansoni parasites were pre-adapted to the Americas and able to establish with relative ease.
Asunto(s)
Biomphalaria , Parásitos , Américas , Animales , Biomphalaria/genética , Biomphalaria/parasitología , Humanos , Schistosoma mansoni/genética , Senegal/epidemiología , Caracoles/genética , TanzaníaRESUMEN
Mesenteric infection by the parasitic blood fluke Schistosoma bovis is a common veterinary problem in Africa and the Middle East and occasionally in the Mediterranean Region. The species also has the ability to form interspecific hybrids with the human parasite S. haematobium with natural hybridisation observed in West Africa, presenting possible zoonotic transmission. Additionally, this exchange of alleles between species may dramatically influence disease dynamics and parasite evolution. We have generated a 374 Mb assembly of the S. bovis genome using Illumina and PacBio-based technologies. Despite infecting different hosts and organs, the genome sequences of S. bovis and S. haematobium appeared strikingly similar with 97% sequence identity. The two species share 98% of protein-coding genes, with an average sequence identity of 97.3% at the amino acid level. Genome comparison identified large continuous parts of the genome (up to several 100 kb) showing almost 100% sequence identity between S. bovis and S. haematobium. It is unlikely that this is a result of genome conservation and provides further evidence of natural interspecific hybridization between S. bovis and S. haematobium. Our results suggest that foreign DNA obtained by interspecific hybridization was maintained in the population through multiple meiosis cycles and that hybrids were sexually reproductive, producing viable offspring. The S. bovis genome assembly forms a highly valuable resource for studying schistosome evolution and exploring genetic regions that are associated with species-specific phenotypic traits.
Asunto(s)
Hibridación Genética/genética , Schistosoma/genética , África , África Occidental , Animales , Secuencia de Bases/genética , Bovinos , Mapeo Cromosómico/métodos , ADN/genética , Genoma/genética , Genoma Mitocondrial/genética , Hibridación Genética/fisiología , Medio Oriente , Filogenia , Proteoma/genética , Especificidad de la Especie , Trematodos/genética , Secuenciación Completa del Genoma/métodosRESUMEN
Do mutations required for adaptation occur de novo, or are they segregating within populations as standing genetic variation? This question is key to understanding adaptive change in nature, and has important practical consequences for the evolution of drug resistance. We provide evidence that alleles conferring resistance to oxamniquine (OXA), an antischistosomal drug, are widespread in natural parasite populations under minimal drug pressure and predate OXA deployment. OXA has been used since the 1970s to treat Schistosoma mansoni infections in the New World where S. mansoni established during the slave trade. Recessive loss-of-function mutations within a parasite sulfotransferase (SmSULT-OR) underlie resistance, and several verified resistance mutations, including a deletion (p.E142del), have been identified in the New World. Here we investigate sequence variation in SmSULT-OR in S. mansoni from the Old World, where OXA has seen minimal usage. We sequenced exomes of 204 S. mansoni parasites from West Africa, East Africa and the Middle East, and scored variants in SmSULT-OR and flanking regions. We identified 39 non-synonymous SNPs, 4 deletions, 1 duplication and 1 premature stop codon in the SmSULT-OR coding sequence, including one confirmed resistance deletion (p.E142del). We expressed recombinant proteins and used an in vitro OXA activation assay to functionally validate the OXA-resistance phenotype for four predicted OXA-resistance mutations. Three aspects of the data are of particular interest: (i) segregating OXA-resistance alleles are widespread in Old World populations (4.29-14.91% frequency), despite minimal OXA usage, (ii) two OXA-resistance mutations (p.W120R, p.N171IfsX28) are particularly common (>5%) in East African and Middle-Eastern populations, (iii) the p.E142del allele has identical flanking SNPs in both West Africa and Puerto Rico, suggesting that parasites bearing this allele colonized the New World during the slave trade and therefore predate OXA deployment. We conclude that standing variation for OXA resistance is widespread in S. mansoni.
Asunto(s)
Resistencia a Medicamentos/genética , Oxamniquina/uso terapéutico , Schistosoma mansoni/efectos de los fármacos , Schistosoma mansoni/genética , Esquistosomicidas/uso terapéutico , Adaptación Fisiológica/genética , Alelos , Animales , Cricetinae , Humanos , Niger , Omán , Polimorfismo de Nucleótido Simple/genética , Ratas , Esquistosomiasis mansoni/tratamiento farmacológico , Senegal , Caracoles/parasitología , TanzaníaRESUMEN
Long non-coding, tandem-repetitive regions in mitochondrial (mt) genomes of many metazoans have been notoriously difficult to characterise accurately using conventional sequencing methods. Here, we show how the use of a third-generation (long-read) sequencing and informatic approach can overcome this problem. We employed Oxford Nanopore technology to sequence genomic DNAs from a pool of adult worms of the carcinogenic parasite, Schistosoma haematobium, and used an informatic workflow to define the complete mt non-coding region(s). Using long-read data of high coverage, we defined six dominant mt genomes of 33.4 kb to 22.6 kb. Although no variation was detected in the order or lengths of the protein-coding genes, there was marked length (18.5 kb to 7.6 kb) and structural variation in the non-coding region, raising questions about the evolution and function of what might be a control region that regulates mt transcription and/or replication. The discovery here of the largest tandem-repetitive, non-coding region (18.5 kb) in a metazoan organism also raises a question about the completeness of some of the mt genomes of animals reported to date, and stimulates further explorations using a Nanopore-informatic workflow.
Asunto(s)
Genoma de los Helmintos , Genoma Mitocondrial , Secuenciación de Nanoporos , Schistosoma haematobium/genética , Secuencias Repetidas en Tándem , AnimalesRESUMEN
In West Africa, Schistosoma spp. are capable of infecting multiple definitive hosts, a lifecycle feature that may complicate schistosomiasis control. We characterized the evolutionary relationships among multiple Schistosoma mansoni isolates collected from snails (intermediate hosts), humans (definitive hosts), and rodents (definitive hosts) in Senegal. On a local scale, diagnosis of S. mansoni infection ranged 3.8%-44.8% in school-aged children, 1.7%-52.6% in Mastomys huberti mice, and 1.8%-7.1% in Biomphalaria pfeifferi snails. Our phylogenetic framework confirmed the presence of multiple S. mansoni lineages that could infect both humans and rodents; divergence times of these lineages varied (0.13-0.02 million years ago). We propose that extensive movement of persons across West Africa might have contributed to the establishment of these various multihost S. mansoni clades. High S. mansoni prevalence in rodents at transmission sites frequented by humans further highlights the implications that alternative hosts could have on future public health interventions.
Asunto(s)
Biomphalaria , Schistosoma mansoni , África Occidental , Animales , Ratones , Filogenia , Schistosoma mansoni/genética , Senegal/epidemiologíaRESUMEN
Introgression among parasite species has the potential to transfer traits of biomedical importance across species boundaries. The parasitic blood fluke Schistosoma haematobium causes urogenital schistosomiasis in humans across sub-Saharan Africa. Hybridization with other schistosome species is assumed to occur commonly, because genetic crosses between S. haematobium and livestock schistosomes, including S. bovis, can be staged in the laboratory, and sequencing of mtDNA and rDNA amplified from microscopic miracidia larvae frequently reveals markers from different species. However, the frequency, direction, age, and genomic consequences of hybridization are unknown. We hatched miracidia from eggs and sequenced the exomes from 96 individual S. haematobium miracidia from infected patients from Niger and the Zanzibar archipelago. These data revealed no evidence for contemporary hybridization between S. bovis and S. haematobium in our samples. However, all Nigerien S. haematobium genomes sampled show hybrid ancestry, with 3.3-8.2% of their nuclear genomes derived from S. bovis, providing evidence of an ancient introgression event that occurred at least 108-613 generations ago. Some S. bovis-derived alleles have spread to high frequency or reached fixation and show strong signatures of directional selection; the strongest signal spans a single gene in the invadolysin gene family (Chr. 4). Our results suggest that S. bovis/S. haematobium hybridization occurs rarely but demonstrate profound consequences of ancient introgression from a livestock parasite into the genome of S. haematobium, the most prevalent schistosome species infecting humans.
Asunto(s)
Introgresión Genética , Proteínas del Helminto/genética , Hibridación Genética , Metaloendopeptidasas/genética , Schistosoma/genética , Animales , Variación Genética , Genoma Mitocondrial , Secuenciación del ExomaRESUMEN
Reliable diagnosis of human helminth infection(s) is essential for ongoing disease surveillance and disease elimination. Current WHO-recommended diagnostic assays are unreliable in low-endemic near-elimination settings and typically involve the invasive, onerous and potentially hazardous sampling of bodily fluids such as stool and blood, as well as tissue via biopsy. In contrast, diagnosis by use of non-invasive urine sampling is generally painless, more convenient and low risk. It negates the need for specialist staff, can usually be obtained immediately upon request and is better accepted by patients. In some instances, urine-based diagnostic assays have also been shown to provide a more reliable diagnosis of infection when compared to traditional methods that require alternative and more invasive bodily samples, particularly in low-endemicity settings. Given these relative benefits, we identify and review current research literature to evaluate whether non-invasive urine sampling is currently exploited to its full potential in the development of diagnostic tools for human helminthiases. Though further development, assessment and validation are needed before their routine use in control programmes, low-cost, rapid and reliable assays capable of detecting transrenal helminth-derived antigens and cell-free DNA show excellent promise for future use at the point-of-care in high-, medium- and even low-endemicity elimination settings.
Asunto(s)
Helmintiasis/diagnóstico , Orina/parasitología , Animales , Antígenos Helmínticos/análisis , Biomarcadores/análisis , ADN de Helmintos/análisis , Proteína Catiónica del Eosinófilo/análisis , Heces/parasitología , Helmintos/aislamiento & purificación , Humanos , Esquistosomiasis/diagnóstico , Esquistosomiasis/patologíaRESUMEN
Accurate diagnosis of urogenital schistosomiasis is crucial for disease surveillance and control. Routine diagnostic methods, however, lack sensitivity when assessing patients with low levels of infection still able to maintain pathogen transmission. Therefore, there is a need for highly sensitive diagnostic tools that can be used at the point-of-care in endemic areas. Recombinase polymerase amplification (RPA) is a rapid and sensitive diagnostic tool that has been used to diagnose several pathogens at the point-of-care. Here, the analytical performance of a previously developed RPA assay (RT-ShDra1-RPA) targeting the Schistosoma haematobium Dra1 genomic region was assessed using commercially synthesised S. haematobium Dra1 copies and laboratory-prepared samples spiked with S. haematobium eggs. Clinical performance was also assessed by comparing diagnostic outcomes with that of a reference diagnostic standard, urine-egg microscopy. The RT-ShDra1-RPA was able to detect 1 × 101 copies of commercially synthesised Dra1 DNA as well as one S. haematobium egg within laboratory-spiked ddH2O samples. When compared with urine-egg microscopy, the overall sensitivity and specificity of the RT-ShDra1-RPA assay was 93.7% (±88.7-96.9) and 100% (±69.1-100), respectively. Positive and negative predictive values were 100% (±97.5-100) and 50% (±27.2-72.8), respectively. The RT-ShDra1-RPA therefore shows promise as a rapid and highly sensitive diagnostic tool able to diagnose urogenital schistosomiasis at the point-of-care.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , Schistosoma haematobium/genética , Esquistosomiasis Urinaria/diagnóstico , Sistema Urogenital/parasitología , Animales , ADN/análisis , Reacciones Falso Positivas , Femenino , Humanos , Sistemas de Atención de Punto , Valor Predictivo de las Pruebas , Recombinasas , Estándares de Referencia , Reproducibilidad de los Resultados , Esquistosomiasis Urinaria/orina , Sensibilidad y Especificidad , Orina/parasitologíaRESUMEN
Molecular analysis of atypical schistosome eggs retrieved from children in Malawi revealed genetic interactions occurring between human (Schistosoma haematobium) and livestock (S. mattheei and S. bovis) schistosome species. Detection of hybrid schistosomes adds a notable new perspective to the epidemiology and control of urogenital schistosomiasis in central Africa.
Asunto(s)
Schistosoma haematobium/clasificación , Schistosoma/clasificación , Esquistosomiasis/epidemiología , Esquistosomiasis/parasitología , Animales , Niño , Humanos , Ganado/parasitología , Malaui/epidemiología , Vigilancia en Salud Pública , Schistosoma/genética , Schistosoma haematobium/genética , Esquistosomiasis/diagnóstico , Esquistosomiasis Urinaria/diagnóstico , Esquistosomiasis Urinaria/epidemiología , Esquistosomiasis Urinaria/parasitologíaRESUMEN
To accelerate the development of novel vaccines for schistosomiasis, we set out to develop a human model for Schistosoma mansoni infection in healthy volunteers. During natural infections, female schistosomes produce eggs that give rise to morbidity. Therefore, we produced single-sex, male Schistosoma mansoni cercariae for human infection without egg production and associated pathology. Cercariae were produced in their intermediate snail hosts in accordance with the principles of good manufacturing practice (GMP). The application of GMP principles to an unconventional production process is a showcase for the controlled production of complex live challenge material in the European Union or under Food and Drug Administration guidance.
Asunto(s)
Schistosoma mansoni/inmunología , Esquistosomiasis mansoni/prevención & control , Esquistosomiasis/prevención & control , Caracoles/parasitología , Animales , Cercarias , Humanos , Masculino , Esquistosomiasis/parasitología , Esquistosomiasis mansoni/parasitologíaRESUMEN
Since the construction of the Diama Dam (1985), the epidemiology of schistosomiasis along the Senegal River Basin (SRB) has been extremely dynamic with outbreaks of both intestinal and urogenital schistosomiasis. In the early 2000s, technicians reported cases of suspected urogenital schistosomiasis in adults from the local hospital in Richard-Toll, Lower SRB. The genetic analysis of schistosome miracidia isolated from 11 patients in 2012 from two neighbourhoods (Campement and Gaya) of Richard-Toll confirmed infection with Schistosoma haematobium but also S. haematobium/S. bovis hybrids. Thirty-seven per cent of the miracidia were S. bovis/S. haematobium hybrids and 63% were pure S. haematobium. The data are discussed in relation to the ongoing dynamic epidemiology of the schistosomes in Senegal and the need to treat non-target individuals.
Asunto(s)
Hibridación Genética , Schistosoma haematobium/genética , Schistosoma/genética , Esquistosomiasis Urinaria/epidemiología , Sistema Urinario/parasitología , Adulto , Animales , Ciclooxigenasa 1/genética , Brotes de Enfermedades , Humanos , Reacción en Cadena de la Polimerasa , Schistosoma/aislamiento & purificación , Schistosoma haematobium/aislamiento & purificación , Esquistosomiasis Urinaria/tratamiento farmacológico , Senegal/epidemiologíaRESUMEN
The causative agent of urogenital schistosomiasis, Schistosoma haematobium, was thought to be the only schistosome species transmitted through Bulinus snails on Unguja and Pemba Island (Zanzibar, United Republic of Tanzania). For insights into the environmental risk of S. haematobium transmission on Pemba Island, malacological surveys collecting Bulinus globosus and B. nasutus, two closely related potential intermediate hosts of S. haematobium were conducted across the island in November 2016. Of 1317 B. globosus/B. nasutus collected, seven B. globosus, identified through sequencing a DNA region of the mitochondrial cytochrome oxidase subunit 1 (cox1), were observed with patent infections assumed to be S. haematobium. However, when the collected cercariae were identified through sequencing a region of the cox1 and the nuclear internal transcribed spacer (ITS1 + 2), schistosomes from five of these B. globosus collected from a single locality were in fact S. bovis. The identified presence of S. bovis raises concerns for animal health on Pemba, and complicates future transmission monitoring of S. haematobium. These results show the pertinence for not only sensitive, but also species-specific markers to be used when identifying cercariae during transmission monitoring, and also provide the first molecular confirmation for B. globosus transmitting S. bovis in East Africa.
Asunto(s)
Bulinus/parasitología , Schistosoma/clasificación , Esquistosomiasis/transmisión , Animales , Cercarias/clasificación , Cercarias/aislamiento & purificación , ADN Intergénico/genética , Complejo IV de Transporte de Electrones/genética , Islas del Oceano Índico/epidemiología , Schistosoma/aislamiento & purificación , Schistosoma haematobium/genética , Schistosoma haematobium/aislamiento & purificación , Esquistosomiasis/epidemiología , Esquistosomiasis Urinaria/epidemiología , Especificidad de la Especie , Tanzanía/epidemiologíaRESUMEN
Adult schistosomes live in the blood vessels and cannot easily be sampled from humans, so archived miracidia larvae hatched from eggs expelled in feces or urine are commonly used for population genetic studies. Large collections of archived miracidia on FTA cards are now available through the Schistosomiasis Collection at the Natural History Museum (SCAN). Here we describe protocols for whole genome amplification of Schistosoma mansoni and Schistosome haematobium miracidia from these cards, as well as real time PCR quantification of amplified schistosome DNA. We used microgram quantities of DNA obtained for exome capture and sequencing of single miracidia, generating dense polymorphism data across the exome. These methods will facilitate the transition from population genetics, using limited numbers of markers to population genomics using genome-wide marker information, maximising the value of collections such as SCAN.
Asunto(s)
Secuenciación del Exoma , Genoma de los Helmintos , Técnicas de Amplificación de Ácido Nucleico , Schistosoma haematobium/genética , Schistosoma mansoni/genética , Animales , Bancos de Muestras Biológicas , Niño , ADN de Helmintos/genética , Heces/parasitología , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Polimorfismo GenéticoRESUMEN
Opisthorchis viverrini is a major public health concern in Southeast Asia. Various reports have suggested that this parasite may represent a species complex, with genetic structure in the region perhaps being dictated by geographical factors and different species of intermediate hosts. We used four microsatellite loci to analyze O. viverrini adult worms originating from six species of cyprinid fish in Thailand and Lao PDR. Two distinct O. viverrini populations were observed. In Ban Phai, Thailand, only one subgroup occurred, hosted by two different fish species. Both subgroups occurred in fish from That Luang, Lao PDR, but were represented to very different degrees among the fish hosts there. Our data suggest that, although geographical separation is more important than fish host specificity in influencing genetic structure, it is possible that two species of Opisthorchis, with little interbreeding, are present near Vientiane in Lao PDR.
Asunto(s)
Cyprinidae/parasitología , Enfermedades de los Peces/parasitología , Opistorquiasis/veterinaria , Opisthorchis/genética , Animales , Enfermedades de los Peces/epidemiología , Humanos , Laos/epidemiología , Repeticiones de Microsatélite/genética , Opistorquiasis/epidemiología , Opistorquiasis/parasitología , HumedalesRESUMEN
BACKGROUND: Urogenital schistosomiasis is caused by the parasitic trematode Schistosoma haematobium. Sensitive and specific point-of-care diagnostics are needed for elimination of this disease. Recombinase polymerase amplification (RPA) assays meet these criteria, and an assay to diagnose S. haematobium has been developed (Sh-RPA). However, false-positive results can occur, and optimisation of reaction conditions to mitigate these is needed. Ease of use and compatibility of DNA extraction methods must also be considered. METHODS: Using synthetic DNA, S. haematobium genomic DNA (gDNA), and urine samples from clinical cases, Sh-RPA reactions incorporating different betaine concentrations (0 M, 1 M, 2.5 M, 12.5 M) and the sample-to-water ratios were tested to determine effects on assay specificity and sensitivity. In addition, five commercial DNA extraction kits suitable for use in resource-limited settings were used to obtain gDNA from single S. haematobium eggs and evaluated in terms of DNA quality, quantity, and compatibility with the Sh-RPA assay. All samples were also evaluated by quantitative polymerase chain reaction (qPCR) to confirm DNA acquisition. RESULTS: The analytical sensitivity of the Sh-RPA with all betaine concentrations was ≥ 10 copies of the synthetic Dra1 standard and 0.1 pg of S. haematobium gDNA. The addition of betaine improved Sh-RPA assay specificity in all reaction conditions, and the addition of 2.5 M of betaine together with the maximal possible sample volume of 12.7 µl proved to be the optimum reaction conditions. DNA was successfully isolated from a single S. haematobium egg using all five commercial DNA extraction kits, but the Sh-RPA performance of these kits varied, with one proving to be incompatible with RPA reactions. CONCLUSIONS: The addition of 2.5 M of betaine to Sh-RPA reactions improved reaction specificity whilst having no detrimental effect on sensitivity. This increases the robustness of the assay, advancing the feasibility of using the Sh-RPA assay in resource-limited settings. The testing of commercial extraction kits proved that crude, rapid, and simple methods are sufficient for obtaining DNA from single S. haematobium eggs, and that these extracts can be used with Sh-RPA in most cases. However, the observed incompatibility of specific kits with Sh-RPA highlights the need for each stage of a molecular diagnostic platform to be robustly tested prior to implementation.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico , Sistemas de Atención de Punto , Schistosoma haematobium , Esquistosomiasis Urinaria , Sensibilidad y Especificidad , Animales , Schistosoma haematobium/genética , Schistosoma haematobium/aislamiento & purificación , Esquistosomiasis Urinaria/diagnóstico , Esquistosomiasis Urinaria/orina , Esquistosomiasis Urinaria/parasitología , Técnicas de Amplificación de Ácido Nucleico/métodos , Humanos , ADN de Helmintos/genética , ADN de Helmintos/aislamiento & purificación , Recombinasas/metabolismo , Recombinasas/genética , Técnicas de Diagnóstico Molecular/métodosRESUMEN
BACKGROUND: Lymnaeid snails of the genus Austropeplea are an important vector of the liver fluke (Fasciola hepatica), contributing to livestock production losses in Australia and New Zealand. However, the species status within Austropeplea is ambiguous due to heavy reliance on morphological analysis and a relative lack of genetic data. This study aimed to characterise the mitochondrial genome of A. cf. brazieri, an intermediate host of liver fluke in eastern Victoria. METHODS: The mitochondrial genome was assembled and annotated from a combination of second- and third-generation sequencing data. For comparative purposes, we performed phylogenetic analyses of the concatenated nucleotide sequences of the mitochondrial protein-coding genes, cytochrome c oxidase subunit 1 and 16S genes. RESULTS: The assembled mt genome was 13,757 base pairs and comprised 37 genes, including 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The mt genome length, gene order and nucleotide compositions were similar to related species of lymnaeids. Phylogenetic analyses of the mt nucleotide sequences placed A. cf. brazieri within the same clade as Orientogalba ollula with strong statistical supports. Phylogenies of the cox1 and 16S mt sequences were constructed due to the wide availability of these sequences representing the lymnaeid taxa. As expected in both these phylogenies, A. cf. brazieri clustered with other Austropeplea sequences, but the nodal supports were low. CONCLUSIONS: The representative mt genome of A. cf. brazieri should provide a useful resource for future molecular, epidemiology and parasitological studies of this socio-economically important lymnaeid species.
Asunto(s)
Genoma Mitocondrial , Filogenia , Caracoles , Animales , Genoma Mitocondrial/genética , Caracoles/parasitología , Australia , Fasciola hepatica/genética , Fasciola hepatica/clasificación , Complejo IV de Transporte de Electrones/genética , Vectores de Enfermedades , Análisis de Secuencia de ADNRESUMEN
Schistosomiasis is a neglected tropical disease (NTD) caused by infection with parasitic trematodes of the genus Schistosoma that can lead to debilitating morbidity and mortality. The World Health Organization recommend molecular xenomonitoring of Biomphalaria spp. freshwater snail intermediate hosts of Schistosoma mansoni to identify highly focal intestinal schistosomiasis transmission sites and monitor disease transmission, particularly in low-endemicity areas. A standardised protocol to do this, however, is needed. Here, two previously published primer sets were selected to develop and validate a multiplex molecular xenomonitoring end-point PCR assay capable of detecting S. mansoni infections within individual Biomphalaria spp. missed by cercarial shedding. The assay proved highly sensitive and highly specific in detecting and amplifying S. mansoni DNA and also proved highly sensitive in detecting and amplifying non-S. mansoni trematode DNA. The optimised assay was then used to screen Biomphalaria spp. collected from a S. mansoni-endemic area for infection and successfully detected S. mansoni infections missed by cercarial shedding as well as infections with non-S. mansoni trematodes. The continued development and use of molecular xenomonitoring assays such as this will aid in improving disease control efforts, significantly reducing disease-related morbidities experienced by those in schistosomiasis-endemic areas.