Population genetics of African Schistosoma species.

Blood flukes within the genus Schistosoma (schistosomes) are responsible for the major disease, schistosomiasis, in tropical and sub-tropical areas. This disease is predominantly present on the African continent with more than 85% of the human cases. Schistosomes are also parasites of veterinary importance infecting livestock and wildlife. Schistosoma population genetic structure and diversity are important characteristics that may reflect variations in selection pressures such as those induced by host (mammalian and snail) environments, habitat change, migration and also treatment/control interventions, all of which also shape speciation and evolution of the whole Schistosoma genus. Investigations into schistosome population genetic structure, diversity and evolution has been an area of important debate and research. Supported by advances in molecular techniques with capabilities for multi-locus genetic analyses for single larvae schistosome genetic investigations have greatly progressed in the last decade. This paper aims to review the genetic studies of both animal and human infecting schistosome. Population genetic structures are reviewed at different spatial scales: local, regional or continental (i.e. phylogeography). Within species genetic diversities are discussed compared and the compounding factors discussed, including the effect of mass drug administration. Finally, the ability for intra-species hybridisation questions species integrities and poses many questions in relation to the natural epidemiology of co-endemic species. Here we review molecularly confirmed hybridisation events (in relation to human disease) and discuss the possible impact for ongoing and future control and elimination.

COMMUNITY CO-DESIGNED SCHISTOSOMIASIS CONTROL INTERVENTIONS FOR SCHOOL-AGED CHILDREN IN ZANZIBAR.

Top-down biomedical interventions to control schistosomiasis in sub-Saharan Africa have had limited success, primarily because they fail to engage with the social, political, economic and ecological contexts in which they are delivered. Despite the call to foster community engagement and to adapt interventions to local circumstances, programmes have rarely embraced such an approach. This article outlines a community co-designed process, based upon Human-Centered Design, to demonstrate how this approach works in practice. It is based on initial work undertaken by social science researchers, public health practitioners and community members from the Zanzibar Islands, Tanzania, between November 2011 and December 2013. During the process, 32 community members participated in a qualitative and quantitative data-driven workshop where they interpreted data on local infections from S. haematobium and co-designed interventions with the assistance of a facilitator trained in the social sciences. These interventions included the implementation of novel school-based education and training, the identification of relevant safe play activities and events at local schools, the installation of community-designed urinals for boys and girls and the installation of community-designed laundry-washing platforms to reduce exposure to cercariae-contaminated fresh water. It is suggested that the a community co-designed process, drawing from Human-Centered Design principles and techniques, enables the development of more sustainable and effective interventions for the control of schistosomiasis.

HEALTH EDUCATION AND THE CONTROL OF UROGENITAL SCHISTOSOMIASIS: ASSESSING THE IMPACT OF THE JUMA NA KICHOCHO COMIC-STRIP MEDICAL BOOKLET IN ZANZIBAR.

Endeavours to control urogenital schistosomiasis on Unguja Island (Zanzibar) have focused on school-aged children. To assess the impact of an associated health education campaign, the supervised use of the comic-strip medical booklet Juma na Kichocho by Class V pupils attending eighteen primary schools was investigated. A validated knowledge and attitudes questionnaire was completed at baseline and repeated one year later following the regular use of the booklet during the calendar year. A scoring system (ranging from 0.0 to 5.0) measured children's understandings of schistosomiasis and malaria, with the latter being a neutral comparator against specific changes for schistosomiasis. In 2006, the average score from 751 children (328 boys and 423 girls) was 2.39 for schistosomiasis and 3.03 for malaria. One year later, the score was 2.43 for schistosomiasis and 2.70 for malaria from 779 children (351 boys and 428 girls). As might be expected, knowledge and attitudes scores for schistosomiasis increased (+0.05), but not as much as originally hoped, while the score for malaria decreased (-0.33). According to a Kolmogorov-Smirnov test, neither change was statistically significant. Analysis also revealed that 75% of school children misunderstood the importance of reinfection after treatment with praziquantel. These results are disappointing. They demonstrate that it is mistaken to assume that knowledge conveyed in child-friendly booklets will necessarily be interpreted, and acted upon, in the way intended. If long-term sustained behavioural change is to be achieved, health education materials need to engage more closely with local understandings and responses to urogenital schistosomiasis. This, in turn, needs to be part of the development of a more holistic, biosocial approach to the control of schistosomiasis.

The Establishment and Function of Schistosomiasis Surveillance System Towards Elimination in The People's Republic of China.

Schistosoma japonicum is the main schistosome species in The People's Republic of China, causing intestinal schistosomiasis, a debilitating disease of public health importance. The People's Republic of China used to be heavily endemic with schistosomiasis, but great progress has been made through the vigorous efforts of the national control programmes in the last six decades. Presently, efforts are geared towards eliminating schistosomiasis from The People's Republic of China by the end of 2025 through effective schistosomiasis surveillance, an important component in the drive towards schistosomiasis elimination. Therefore, this article explicitly outlines the development and progress made in schistosomiasis surveillance since 1990 with a special focus on the new surveillance system in use. Although the surveillance system has steadily improved over the years, it is faced with many challenges. Hence, more efforts are needed to establish an effective and sensitive evaluation system for the national schistosomiasis elimination programme in The People's Republic of China.

Erratum to: Development of novel multiplex microsatellite polymerase chain reactions to enable high-throughput population genetic studies of Schistosoma haematobium.

Unfortunately, the original version of this article [1], contained a mistake. In Table 1, the primers for Sh6 and Sh9 were included incorrectly. Instead of GGGATGTATGCAGACTTG TTGTTTGGCTGCAGTAAC and GCTGAGCTTGAGATTG CTTCTGTCCCATCGATACC they should have been Sh6 Forward Primer GGTGGATTACGCAATAG, Sh6 Reverse Primer TTTAATCAACCGGGTGTC and Sh9 Forward Primer GGGATGTATGCAGACTTG, Sh9 Reverse Primer TTGTTTGGCTGCAGTAAC respectively. A corrected version of Table 1 is included below

Development of novel multiplex microsatellite polymerase chain reactions to enable high-throughput population genetic studies of Schistosoma haematobium.

BACKGROUND: Human urogenital schistosomiasis caused by Schistosoma haematobium is widely distributed across Africa and is increasingly targeted for control and regional elimination. The development of new high-throughput, cost-effective molecular tools and approaches are needed to monitor and evaluate the impact of control programs on the parasite populations. Microsatellite loci are genetic markers that can be used to investigate how parasite populations change over time and in relation to external influences such as control interventions. FINDINGS: Here, 18 existing S. haematobium microsatellite loci were optimised to enable simultaneous amplification across two novel multiplex microsatellite PCR's, each containing nine loci. Methods were developed for the cost effective and rapid processing and microsatellite analysis of S. haematobium larval stages stored on Whatman-FTA cards and proved robust on miracidia and cercariae collected from Zanzibar and Niger. CONCLUSION: The development of these novel and robust multiplex microsatellite assays, in combination with an improved protocol to elute gDNA from Whatman-FTA fixed schistosome larval stages, enables the high-throughput population genetic analysis of S. haematobium. The molecular resources and protocols described here advance the way researchers can perform multi locus-based population genetic analyses of S. haematobium as part of the evaluation and monitoring of schistosomiasis control programmes.

Isothermal Recombinase Polymerase amplification (RPA) of Schistosoma haematobium DNA and oligochromatographic lateral flow detection.

BACKGROUND: Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs. Amplification of schistosome DNA in urine by PCR is sensitive and specific but requires infrastructure, financial resources and skilled personnel, often not available in endemic areas. Recombinase Polymerase Amplification (RPA) is an isothermal DNA amplification/detection technology that is simple, rapid, portable and needs few resources. FINDINGS: Here a Schistosoma haematobium RPA assay was developed and adapted so that DNA amplicons could be detected using oligochromatographic Lateral Flow (LF) strips. The assay successfully amplified S. haematobium DNA at 30-45 °C in 10 mins and was sensitive to a lower limit of 100 fg of DNA. The assay was also successful with the addition of crude urine, up to 5% of the total reaction volume. Cross amplification occurred with other schistosome species but not with other common urine microorganisms. CONCLUSION: The LF-RPA assay developed here can amplify and detect low levels of S. haematobium DNA. Reactions are rapid, require low temperatures and positive reactions are interpreted using lateral flow strips, reducing the need for infrastructure and resources. This together with an ability to withstand inhibitors within urine makes RPA a promising technology for further development as a molecular diagnostic tool for urogenital schistosomiasis.

Regular treatments of praziquantel do not impact on the genetic make-up of Schistosoma mansoni in Northern Senegal.

The Senegal River Basin (SRB) experienced a major epidemic of intestinal schistosomiasis in the early nineties, after the construction of a dam for irrigation purposes. Exceptionally low cure rates following praziquantel (PZQ) treatment at the onset of the epidemic raised concerns about PZQ resistant strains of Schistosoma mansoni, although they could also be attributed to the intense transmission at that time. A field study in the same region more than 15 years later found cure rates for S. mansoni still to be low, whereas Schistosomahaematobium responded well to treatment. We collected S. mansoni miracidia from children at base-line prior to treatment, six months after two PZQ treatments and two years after the start of the study when they had received a total of five PZQ treatments. In total, 434 miracidia from 12 children were successfully genotyped with at least six out of nine DNA microsatellite loci. We found no significant differences in the genetic diversity of, and genetic differentiation between parasite populations before and after repeated treatment, suggesting that PZQ treatment does not have an impact on the neutral evolution of the parasite. This is in stark contrast with a similar study in Tanzania where a significant decrease in genetic diversity was observed in S. mansoni miracidia after a single round of PZQ treatment. We argue that PZQ resistance might play a role in our study area, although rapid re-infection cannot be excluded. It is important to monitor this situation carefully and conduct larger field studies with short-term follow-up after treatment. Since PZQ is the only general schistosomicide available, the possibility of PZQ resistance is of great concern both for disease control and for curative use in clinical practice.

Population genetics of Schistosoma haematobium: development of novel microsatellite markers and their application to schistosomiasis control in Mali.

The recent implementation of mass drug administration (MDA) for control of uro-genital schistosomiasis has identified an urgent need for molecular markers to both directly monitor the impact of MDA, for example to distinguish re-infections from uncleared infections, as well as understand aspects of parasite reproduction and gene flow which might predict evolutionary change, such as the development and spread of drug resistance. We report the development of a novel microsatellite tool-kit allowing, for the first time, robust genetic analysis of individual S. haematobium larvae collected directly from infected human hosts. We genotyped the parasite populations of 47 children from 2 schools in the Ségou region of Mali, the first microsatellite study of this highly neglected parasite. There was only limited evidence of population subdivision between individual children or between the two schools, suggesting that few barriers to gene flow exist in this population. Complex relationships between parasite reproductive success, infection intensity and host age and gender were identified. Older children and boys harboured more diverse infections, as measured by the number of unique adult genotypes present. Individual parasite genotypes had variable reproductive success both across hosts, a pre-requisite for evolutionary selection, and, phenotypically, in hosts of different ages and genders. These data serve as a baseline against which to measure the effect of treatment on parasite population genetics in this region of Mali, and the tools developed are suitable to further investigate this important pathogen, and its close relatives, throughout their range.

Molecular approaches to the identification of Bulinus species in south-west Nigeria and observations on natural snail infections with schistosomes.

The current study considers the distribution of a small sample of 138 Bulinus snails, across 28 localities within eight Nigerian states. Snails were identified using a combination of molecular methods involving both DNA sequencing of a partial cytochrome oxidase subunit 1 (cox1) fragment and restriction profiles obtained from ribosomal internal transcribed spacer (its) amplicons. The results showed that the majority of Bulinus samples tested belonged to the species Bulinus truncatus while only two were Bulinus globosus. The use of RsaI restriction endonuclease to cleave the ribosomal its of Bulinus, as a method of species identification, was adopted for the majority of samples, this being a quicker and cheaper method better suited to small laboratory environments. Polymerase chain reaction (PCR) amplification of the schistosome Dra1 repeat within each of the collected Bulinus samples was employed to determine the extent and distribution of infected snails within the sample areas. Successful amplification of the Dra1 repeat demonstrated that 29.7% of snails were infected with schistosomes. Sequencing of the partial schistosome its from a small subset of snail samples suggested that some snails were either penetrated by both Schistosoma haematobium and Schistosoma bovis miracidia or hybrid miracidia formed from the two species.

Transmission studies of intestinal schistosomiasis in Lake Albert, Uganda and experimental compatibility of local Biomphalaria spp.

Despite ongoing preventive chemotherapy campaigns, intestinal schistosomiasis is hyper-endemic in shoreline communities living along Lake Albert, Uganda. To provide a deeper insight into the local epidemiology of Schistosoma mansoni, a variety of field-based studies were undertaken focusing upon schistosome-snail interactions and confirmation of transmission foci. Cercarial shedding patterns of field-caught Biomphalaria spp., as identified by morphology, were hourly observed over a ten day period and showed that Biomphalaria stanleyi produced significantly more cercariae than Biomphalaria sudanica. Peak production times in both species were between 12.00 and 14.00h indicating greatest infection risk from lake water exposure is during the early afternoon. Laboratory-bred snails were exposed to locally hatched miracidia and susceptibility of Biomphalaria spp. was confirmed experimentally. Biomphalaria stanleyi was a more permissive host. After ascertaining appropriate conditions for infection of laboratory mice, 28 groups of between 5 and 6 naïve mice were placed in floatation cages at four suspected shoreline transmission sites for a 30 minute period of exposure. Eight weeks later, mice (n=142) were culled and S. mansoni adult worms were retrieved from 10 animals. Taken as a whole, these observations highlight the local importance of B. stanleyi in transmission of intestinal schistosomiasis and clearly demonstrate the risk of infection on the Lake Albert shoreline. To mitigate this risk local environmental modification(s), i.e. improvement in sanitation and hygiene and control of snail populations, is needed to bolster the impact of chemotherapy-based interventions.

Rapid diagnostic multiplex PCR (RD-PCR) to discriminate Schistosoma haematobium and S. bovis.

Schistosoma haematobium and S. bovis are widespread schistosome species causing human and cattle schistosomiasis, respectively, in Africa. The sympatric occurrence of these two species and their ability to infect the same Bulinus intermediate snail hosts necessitates precise methods of identification of the larval stages. A rapid diagnostic 'mulitplex' one-step polymerase chain reaction protocol (RD-PCR) was developed using cytochrome oxidase subunit 1 (COX1) mitochondrial DNA (mtDNA) to discriminate between S. haematobium and S. bovis. A single forward primer and two species-specific reverse primers were used to produce a polymerase chain reaction (PCR) fragment of 306 bp and 543 bp for S. bovis and S. haematobium, respectively. Serial dilutions were carried out on various lifecycle stages and species combinations to test the sensitivity and specificity of the primers. This RD-PCR proved highly sensitive, detecting a single larval stage and as little as 0.78 ng of genomic DNA (gDNA) from an adult schistosome, providing a cost-effective, rapid and robust molecular tool for high-throughput screening of S. haematobium and S. bovis populations. In areas where human and cattle schistosomiasis overlap and are transmitted in close proximity, this mitochondrial assay will be a valuable identification tool for epidemiological studies, especially when used in conjunction with other nuclear diagnostic markers.

Low genetic diversity in a snail intermediate host (Biomphalaria pfeifferi Krass, 1848) and schistosomiasis transmission in the Senegal River Basin.

Population genetic perturbations of intermediate hosts, often a consequence of human pressure on environmental resources, can precipitate unexpectedly severe disease outbreaks. Such disturbances are set to become increasingly common following range changes concomitant with climate shifts, dwindling natural resources and major infrastructure changes such as hydroprojects. Construction of the Diama dam in the Senegal River Basin (SRB) reduced river salinity, enabling the freshwater snail intermediate host Biomphalaria pfeifferi to rapidly expand its distribution. A serious public health problem ensued, with an epidemic of intestinal schistosomiasis occurring in the previously schistosome-free Richard-Toll region within 2 years. The current study aimed to assess the population variability of B. pfeifferi in the SRB, and speculate upon its subsequent impact on host-parasite interactions following such engineered ecological change. Genetic variation at nine polymorphic microsatellite loci revealed little population differentiation in SRB snails compared with those from natural habitats in Zimbabwe, where Schistosoma mansoni transmission is much lower. 'Open' SRB habitats are associated with greater water contact, smaller population sizes and less genetic diversity, with sites downstream of Richard-Toll showing greater inter- and intrapopulation variation, concomitant with less frequent human contact. These observations may be explained by rapid expansion into pristine habitat selecting for high fecundity genotypes at the expense of schistosome resistance, presenting S. mansoni with genetically homogenous highly fecund susceptible populations around the focal point, promoting development of a highly compatible host-parasite relationship. Longitudinal study of such systems may prove important in predicting public health risks engendered by future environmental engineering projects.

Molecular epidemiology of Schistosoma mansoni in Uganda: DNA barcoding reveals substantial genetic diversity within Lake Albert and Lake Victoria populations.

Representative samples of Ugandan Schistosoma mansoni from Lake Albert and Lake Victoria were examined using DNA barcoding, sequence analysis of two partially overlapping regions - ASMIT (396 bp) & MORGAN (617 bp) - of the mitochondrial cytochrome oxidase subunit I (cox1). The Victorian sample exhibited greater nucleotide diversity, 1.4% vs. 1.0%, and a significant population partition appeared as barcodes did not cross-over between lakes. With one exception, Lake Albert populations were more mixed by sampled location, while those from Lake Victoria appeared more secluded. Using statistical parsimony, barcode ASMIT 1 was putatively ancestral to all others and analysis of MORGAN cox1 confirmed population diversity. All samples fell into two of five well-resolved lineages; sub-lineages therein broadly partitioning by lake. It seems that barcode ASMIT 1 (and close variants) was likely widely dispersed throughout the Nilotic environment but later diversified in situ, and in parallel, within Lake Albert and Lake Victoria. The genetic uniformity of Ugandan S. mansoni can no longer be assumed, which might better explain known epidemiological heterogeneities. While it appears plausible that locally evolved heritable traits could spread through most of the Lake Albert populations, it seems unlikely they could quickly homogenise into Lake Victoria or amongst populations therein.

A wake up call for urinary schistosomiasis: reconciling research effort with public health importance.

This review considers the current status of urinary schistosomiasis, caused by infection with Schistosoma haematobium, and argues that greater research effort and focus are needed to improve understanding of this neglected tropical disease (NTD). The inappropriateness of relying solely on data concerning the much more extensively studied intestinal form of schistosomiasis caused by S. mansoni is highlighted. The current lack of genome and transcriptome information for S. haematobium is directly hindering further targeted research and must be quickly rectified. Recent molecular phylogenies caution the expectation of similarities between schistosome species and highlight the close relationships of species within the S. haematobium group. Treatment, current and prospective drugs and vaccines, together with diagnosis are considered, highlighting the differences associated with urinary schistosomiasis. This infection has a significant and specific impact on the urino-genital system and has a strong association with bladder cancer, leading to severe and chronic morbidity. There is a clear need for new clinical initiatives in this area to better quantify the disease burden. Furthermore, emerging associations with HIV and other pathogens need to be closely monitored. Research is urgently needed to improve current knowledge in order to develop the next generation of control tools.

Host choice and penetration by Schistosoma haematobium miracidia.

Schistosome parasites commonly show specificity to their intermediate mollusc hosts and the degree of specificity can vary between parasite strains and geographical location. Here the role of miracidial behaviour in host specificity of Schistosoma haematobium on the islands of Zanzibar is investigated. In choice-chamber experiments, S. haematobium miracidia moved towards Bulinus globosus snail hosts in preference to empty chambers. In addition, miracidia preferred uninfected over patent B. globosus. This preference should benefit the parasite as patent snails are likely to have mounted an immune response to S. haematobium as well as providing poorer resources than uninfected snails. Miracidia also discriminated between the host B. globosus and the sympatric, non-host species Cleopatra ferruginea. In contrast, S. haematobium did not discriminate against the allopatric Bulinus nasutus. Penetration of the host by miracidia was investigated by screening snails 24 h after exposure using polymerase chain reaction (PCR) with S. haematobium specific DraI repeat primers. There was no difference in the frequency of penetration of B. globosus versus B. nasutus. These responses to different snail species may reflect selection pressure to avoid sympatric non-hosts which represent a transmission dead end. The distribution of B. nasutus on Unguja is outside the endemic zone and so there is less chance of exposure to S. haematobium, hence there will be little selection pressure to avoid this non-host snail.