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Schistosomiasis is a parasitic disease affecting over 200 million people worldwide. This study reports the design and development of a microfiltration device for isolating schistosome eggs in urine for rapid diagnostics of urogenital schistosomiasis. The design of the device comprises a linear array of microfluidic traps to immobilize and separate schistosome eggs. Sequential loading of individual eggs is achieved autonomously by flow resistance, which facilitates observation and enumeration of samples with low-abundance targets. Computational fluid dynamics modeling and experimental characterization are performed to optimize the trapping performance. By optimizing the capture strategy, the trapping efficiency could be achieved at 100% with 300 μl/min and 83% with 3000 μl/min, and the filtration procedure could be finished within 10 min. The trapped eggs can be either recovered for downstream analysis or preserved in situ for whole-mount staining. On-chip phenotyping using confocal laser fluorescence microscopy identifies the microstructure of the trapped schistosome eggs. The device provides a novel microfluidic approach for trapping, counting and on-chip fluorescence characterization of urinal Schistosoma haematobium eggs for clinical and investigative application.

Scientists and clinicians studying a particular disease have an ideal goal that, if achieved, would be paradoxical: finding the disease cure and thereby putting themselves out of work. 2015 marks the 35th year since the cure for smallpox eradicated this human scourge. Before a vaccine was developed, infection with smallpox virus occurred in over 10 million people per year around the world, with a death rate greater than 30% [1]. The World Health Organization’s (WHO’s) intensive eradication program against smallpox began in 1967 and was maintained for over ten years. The last known case of smallpox was recorded in Somalia in 1977, and WHO declared the global eradication of smallpox a success in 1980. Vaccinia virus (VACV), like smallpox a member of the Poxviridae family, was used to produce the vaccine that led to smallpox eradication. Today, VACV remains a critical research tool and serves as the laboratory model for poxvirus. VACV is currently used for the production of 2nd- and 3rd-generation smallpox vaccines as well as for the exploration of immuno-oncolytic therapies against melanoma and other cancers [2,3]. VACV and its derivatives, including modified vaccinia virus (MVA) and New York attenuated vaccinia virus (NYVAC), are used as vectors by which to induce immunity to pathogens including HIV, Plasmodium falciparum, and Mycobacterium tuberculosis [4,5]. VACV also continues to be an important tool for examining fundamental viral mechanisms of immune evasion, including viral immunomodulation and inhibition of cell apoptosis, and has aided investigators in characterizing the innate and adaptive host immune response to viral infection [6]. Therefore, despite the eradication of human disease caused by smallpox virus, basic and applied research using the model vaccinia virus continues to guide our understanding of host immune responses, antigen immunogenicity, viral manipulation of host defenses, and vaccine efficacy. A second infectious pathogen that is targeted for eradication is Dracunculus medinensis, or guinea worm, the nematode parasite that causes drancunculiasis. D. medinensis is contracted by drinking larvae-contaminated water. The parasite matures to an adult worm in the host gut and emerges painfully months later through the skin of the lower extremities. Through efforts of the World Health Assembly (as part of WHO), the Carter Center, and other organizations, dracunculiasis is nearing worldwide eradication. In the 1980s, 20 endemic countries accounted for 3.5 million cases of dracunculiasis; by 2014, only 126 cases were reported in four countires: Chad, Ethiopia, Mali, and South Sudan [7,8]. Despite being close to eradicating D. medinensis, we continue to use this parasite to understand endosymbiotic relationships in organisms. Wolbachia, a bacterial symbiont found in many filarial nematodes and necessary for worm survival, are absent in D. medinensis [9]. Understanding why most filarial nematodes require Wolbachia, through comparative laboratory-based studies of Drancunculus, may inform endosymbiont-targeted efforts to eradicate other filarial parasites. Thus, we contend that there is an important scientific role for maintenance of selected pathogens that have otherwise been eradicated, or nearly eradicated, outside the laboratory setting. Like dracunculiasis, schistosomiasis is a tropical disease caused by a parasitic worm. This snail-borne disease is deeply tied to modifiable environmental conditions (i.e., bodies of water conducive to snail survival), a feature which has, in large part, led to calls to set an agenda for schistosomiasis elimination [10]. Schistosomes are spread by freshwater snails when the larval form of schistosomes, the cercariae, emerges from infected snails and penetrates the skin of its human host, taking up residence in blood vessels following maturation to adult worms. The adult worms produce hundreds of eggs per day, some exiting the host in the urine and/or feces, contributing to the pernicious cycle of environmental contamination and reinfection in people. However, many of the eggs become trapped in host tissues including the liver, intestine, and bladder. The damage caused by chronic parasite egg deposition is significant and contributes to the high morbidity and mortality observed in schistosomiasis. Schistosome infection presents unique challenges to worldwide eradication goals and has even proven difficult to eliminate from a targeted endemic region. If eradication of schistosomiasis were achieved, however, the benefits of schistosomiasis research would remain. In fact, the study of schistosomes, schistosomiasis pathogenesis, and immunological characterizations of the host response to parasite egg antigen are currently large areas of study that have been driven by use of schistosomes as a model organism to study stem cell biology, acute and chronic inflammation, liver and gut fibrosis, and T helper 2-type responses, among many others. These and other research objectives will remain a focus of study for many scientists and clinicians, independently of a “cure” for schistosomiasis. For example, it has long been observed that schistosomes persist for years to decades in the blood vessels of humans. This successful parasitism underscores Schistosoma species’ aptitude for host immune evasion and longevity. In recognition of this interesting property of Schistosoma worms, planaria scientists have begun adapting their versatile toolbox to schistosomes, also a flatworm of the phylum Platyhelminthes. Collins et al. [11] have characterized a proliferating somatic cell (PSC) population in adult worms that may contribute to the ability of schistosomes to repair injury and resist senescence. Studying the stem cell biology of long-lived metazoans may reveal pathways relevant to human aging. Schistosomes have also proven to be powerful tools by which some of the earliest delineation of the TH1 versus TH2 immunity paradigm was shaped [12], with implications for immunological pathway discovery across many diseases. Infection with pathogens that induce TH1 CD4+ lymphocytes and secrete interferon gamma (IFNg) and interleukin-2 (IL-2) can be compared to induction of the TH2 response following schistosome infection or exposure to parasite eggs or egg antigen resulting in the production of IL-4, IL-5, and IL-13. In addition, the mouse model of Schistosoma mansoni infection has helped characterize the role of IL-10 as an anti-inflammatory cytokine required to control liver pathology following egg deposition and, more broadly, has helped define IL-10 immunoregulation in other diseases in which inflammation drives pathology, including ulcerative colitis, Crohn’s disease, and, in the lung, allergy and asthma. In addition to schistosomiasis, IL-10 helps control the immunopathogenesis observed following infection with Plasmodium spp., Mycobacterium spp., and Trypanosoma cruzi. Several investigators have taken advantage of the strong immunogenic properties and ease of use of S. mansoni parasite eggs and soluble egg antigens (SEA). Graham et al. [13] have modeled pulmonary hypertension following intravenous injection of Schistosoma eggs and showed that vascular endothelial growth factor (VEGF) contributes to the TH2 environment that supports airway remodeling and vascular inflammation. SEA has also been utilized as an adjuvant to boost a cytotoxic lymphocyte population following vaccination with an HIV-1 Gag construct [14]. Last, and not least, we introduce Schistosoma haematobium to dissect the link between inflammation and cancer. The International Agency for Research in Cancer (IARC) of WHO has declared S. haematobium to be a class I carcinogen for the bladder, "definitely carcinogenic for humans" [15]. Despite the strong link between S. haematobium, the causative agent of urogenital schistosomiasis, and bladder carcinogenesis, we understand very little regarding how this helminth causes cancer. Most hypotheses suggest that S. haematobium-induced chronic inflammation is critical in the oncogenic process. Interestingly, S. haematobium is linked to higher rates of squamous cell carcinoma of the bladder, a form of bladder cancer that is unusual in non-S. haematobium-infected patients. Our inadequate knowledge of schistosomal bladder carcinogenesis underscores the importance of tractable animal models of urogenital schistosomiasis [16–23]. These models have identified potential mediators of fibrosis, immunomodulation, and carcinogenesis that may be common to multiple neoplasms. Given the evidence that urogenital schistosomiasis-induced bladder cancer is an inflammatory process, studying schistosomiasis, even if eradicated, may reveal new links between inflammation and carcinogenesis that are suitable as therapeutic targets in non-schistosomal cancers. Thus, prudent laboratory maintenance and study of particular pathogens that have been globally eliminated or eradicated may yield unique scientific discoveries of broad significance.

Background Over 112 million people worldwide are infected with Schistosoma haematobium, one of the most prevalent schistosome species affecting humans. Female genital schistosomiasis (FGS) occurs when S. haematobium eggs are deposited into the female reproductive tract by adult worms, which can lead to pelvic pain, vaginal bleeding, genital disfigurement and infertility. Recent evidence suggests co-infection with S. haematobium increases the risks of contracting sexually transmitted diseases such as HIV. The associated mechanisms remain unclear due to the lack of a tractable animal model. We sought to create a mouse model conducive to the study of immune modulation and genitourinary changes that occur with FGS. Methods To model FGS in mice, we injected S. haematobium eggs into the posterior vaginal walls of 30 female BALB/c mice. A control group of 20 female BALB/c mice were injected with uninfected LVG hamster tissue extract. Histology, flow cytometry and serum cytokine levels were assessed at 2, 4, 6, and 8 weeks post egg injection. Voiding studies were performed at 1 week post egg injection. Results Vaginal wall injection with S. haematobium eggs resulted in synchronous vaginal granuloma development within 2 weeks post-egg injection that persisted for at least 6 additional weeks. Flow cytometric analysis of vaginal granulomata revealed infiltration by CD4+ T cells with variable expression of the HIV co-receptors CXCR4 and CCR5. Granulomata also contained CD11b+F4/80+ cells (macrophages and eosinophils) as well as CXCR4+MerTK+ macrophages. Strikingly, vaginal wall-injected mice featured significant urinary frequency despite the posterior vagina being anatomically distant from the bladder. This may represent a previously unrecognized overactive bladder response to deposition of schistosome eggs in the vagina. Conclusion We have established a new mouse model that could potentially enable novel studies of genital schistosomiasis in females. Ongoing studies will further explore the mechanisms by which HIV target cells may be drawn into FGS-associated vaginal granulomata., Author Summary Over 112 million people worldwide are infected with Schistosoma haematobium worms. S. haematobium eggs tend to be deposited in the tissue of pelvic organs such as the urinary bladder, lower ureters, cervix and vagina. Key sequelae include hematuria, dysuria, urinary frequency, and an increased risk of bladder cancer. This form of schistosomiasis can also cause dyspareunia, vaginal bleeding, pruritis, and granulomata that appear as tumors in the female genital tract. Collectively, these signs and symptoms are termed female genital schistosomiasis (FGS). Recent studies suggest that FGS occurs more commonly in girls and women with HIV, suggesting that it may be a risk factor for becoming HIV-infected. Unfortunately, the pathophysiology of this co-infection is not well understood. A lack of an experimentally manipulable model has contributed to the paucity of research focusing on this parasite. We have circumvented the barriers to natural S. haematobium oviposition in the mouse by directly microinjecting parasite eggs into the vaginal mucosa. The injection of S. haematobium ova appears to trigger vaginal inflammation and scarring infiltration by leukocytes expressing HIV co-receptors, and increased urinary frequency. Our approach may provide a representative animal model that could contribute to new opportunities to better understand the basic molecular and cellular immunology of female genital schistosomiasis.

Schistosomiasis remains a health burden in many parts of the world. The complex life cycle of Schistosoma parasites and the economic and societal conditions present in endemic areas make the prospect of eradication unlikely in the foreseeable future. Continued and vigorous research efforts must therefore be directed at this disease, particularly since only a single World Health Organization (WHO)-approved drug is available for treatment. The National Institutes of Health (NIH)–National Institute of Allergy and Infectious Diseases (NIAID) Schistosomiasis Resource Center (SRC) at the Biomedical Research Institute provides investigators with the critical raw materials needed to carry out this important research. The SRC makes available, free of charge (including international shipping costs), not only infected host organisms but also a wide array of molecular reagents derived from all life stages of each of the three main human schistosome parasites. As the field of schistosomiasis research rapidly advances, it is likely to become increasingly reliant on omics, transgenics, epigenetics, and microbiome-related research approaches. The SRC has and will continue to monitor and contribute to advances in the field in order to support these research efforts with an expanding array of molecular reagents. In addition to providing investigators with source materials, the SRC has expanded its educational mission by offering a molecular techniques training course and has recently organized an international schistosomiasis-focused meeting. This review provides an overview of the materials and services that are available at the SRC for schistosomiasis researchers, with a focus on updates that have occurred since the original overview in 2008.

Background The Kato-Katz (KK) stool smear is the standard test for the diagnosis of Schistosoma mansoni infection, but suffers from low sensitivity when infections intensities are moderate to low. Thus, misdiagnosed individuals remain untreated and contribute to the disease transmission, thereby forestalling public health efforts to move from a modality of disease control to one of elimination. As an alternative, the urine-based diagnosis of schistosomiasis mansoni via the circulating cathodic antigen immuno-chromatographic test (CCA-ICT) has been extensively evaluated in Africa with the conclusion that it may replace the KK test in areas where prevalences are moderate or high. Methods and Findings The objective was to measure the performance of the CCA-ICT in a sample study population composed of residents from non-endemic and endemic areas for schistosomiasis mansoni in two municipalities of Minas Gerais state, Brazil. Volunteers (130) were classified into three infection status groups based on duplicate Kato-Katz thick smears from one stool sample (2KK test): 41 negative individuals from non-endemic areas, 41 negative individuals from endemic areas and 48 infected individuals from endemic areas. Infection status was also determined by the CCA-ICT and infection exposure by antibody ELISA (enzyme-linked immunosorbent assay) to S. mansoni soluble egg antigen (SEA) and soluble (adult) worm antigen preparation (SWAP). Sensitivity and specificity were influenced by whether the trace score visually adjudicated in the CCA-ICT was characterized as positive or negative for S. mansoni infection. An analysis of a two-graph receiver operating characteristic was performed to change the cutoff point. When the trace score was interpreted as a positive rather than as a negative result, the specificity decreased from 97.6% to 78.0% whereas sensitivity increased from 68.7% to 85.4%. A significantly positive correlation between the CCA-ICT scores and egg counts was identified (r = 0.6252, p = 0.0001). However, the CCA-ICT misdiagnosed as negative 14.6% of 2KK positive individuals, predominantly those with light infections (fewer than 100 eggs/g feces). Considering 2KK as reference test, the discriminating power of the CCA-ICT (the area under the curve [AUC] = 0.817) was greater than the SEA-ELISA (AUC = 0.744) and SWAP-ELISA (AUC = 0.704). Conclusion Our data for the performance of the CCA-ICT in the Brazilian communities endemic for schistosomiasis mansoni support those from Africa, i.e., in areas with greater infection prevalence and intensities, the CCA-ICT may be useful as a tool to indicate community-based preventative chemotherapy without individual diagnosis. However, because of the Brazilian Ministry of Health’s recommendation for individual diagnosis in areas where prevalence is less than 15%, i.e., those areas in which infection intensities are likely to be lowest, the CCA-ICT lacks the sensitivity to be used as standalone diagnostic tool., Author Summary Detecting parasite eggs in stool by the Kato-Katz (KK) stool smear is the standard diagnostic test for infection with the flatworm parasite, Schistosoma mansoni. However, the test can miss those who have low burdens of infection, i.e., with few eggs in their feces. These misdiagnosed individuals, therefore, do not receive drug treatment and can continue to transmit the parasite into the environment putting the community at risk of infection. As an alternative diagnostic approach, the circulating cathodic antigen immuno-chromatographic test (CCA-ICT) is a simple-to-use handheld device (similar to a pregnancy test) that only needs urine to provide a quick and visual indication of whether one is infected or not. The consensus from studies in Africa is that the CCA-ICT could replace the KK test in those areas where people are more likely to harbor moderate to high worm burdens (i.e., more eggs in stool), but, like the KK test, it can miss those harboring light infection intensities. We evaluated the CCA-ICT performance in urine samples from 130 individuals living in areas non-endemic and endemic for schistosomiasis mansoni within the municipalities of Governador Valadares and Manhuaçu, Minas Gerais state, Brazil. The CCA-ICT performance characteristics, chiefly, sensitivity and specificity, depended on whether a ‘trace’ visual reading of the test was considered as a positive or negative diagnosis. We noted a positive correlation between the CCA-ICT scores and egg counts. However, the CCA-ICT misdiagnosed as negative about 15% of KK positive individuals, predominantly those with light infections. The CCA-ICT, nonetheless, had better discriminating power than commonly used antibody-based tests. We conclude that the CCA-ICT offers reasonable performance to diagnosis S. mansoni infection. However, in areas where infections intensities are light, the test lacks the sensitivity to be used as standalone diagnostic tool.

Urogenital schistosomiasis, chronic infection by Schistosoma haematobium, affects 112 million people worldwide. S. haematobium worm oviposition in the bladder wall leads to granulomatous inflammation, fibrosis, and egg expulsion into the urine. Despite the global impact of urogenital schistosomiasis, basic understanding of the associated pathologic mechanisms has been incomplete due to the lack of suitable animal models. We leveraged our recently developed mouse model of urogenital schistosomiasis to perform the first-ever profiling of the early molecular events that occur in the bladder in response to the introduction of S. haematobium eggs. Microarray analysis of bladders revealed rapid, differential transcription of large numbers of genes, peaking three weeks post-egg administration. Many differentially transcribed genes were related to the canonical Type 2 anti-schistosomal immune response, as reflected by the development of egg-based bladder granulomata. Numerous collagen and metalloproteinase genes were differentially transcribed over time, revealing complex remodeling and fibrosis of the bladder that was confirmed by Masson's Trichrome staining. Multiple genes implicated in carcinogenesis pathways, including vascular endothelial growth factor-, oncogene-, and mammary tumor-related genes, were differentially transcribed in egg-injected bladders. Surprisingly, junctional adhesion molecule, claudin and uroplakin genes, key components for maintaining the urothelial barrier, were globally suppressed after bladder exposure to eggs. This occurred in the setting of urothelial hyperplasia and egg shedding in urine. Thus, S. haematobium egg expulsion is associated with intricate modulation of the urothelial barrier on the cellular and molecular level. Taken together, our findings have important implications for understanding host-parasite interactions and carcinogenesis in urogenital schistosomiasis, and may provide clues for novel therapeutic strategies., Author Summary Parasitic Schistosoma haematobium worms cause urogenital schistosomiasis in 112 million people worldwide. These worms lay eggs in the bladder wall, resulting in inflammation, fibrosis (internal scarring), bladder cancer, and passage of eggs into the urine. Indeed, the International Agency for Research on Cancer within the World Health Organization has classified S. haematobium as a “Class I” agent (“Carcinogenic to humans”). Moreover, S. haematobium-induced fibrosis and resulting obstructive kidney failure leads to 150,000 deaths annually. As a result, S. haematobium infection is one of the most important causes of worm-related death globally. In spite of this, research on this parasite is sparse due to a lack of suitable animal models. We have used our recently developed mouse model of urogenital schistosomiasis to understand the global bladder gene response to this infection. Large numbers of genes featured differential transcription after experimental infection, including specific immune response-, fibrosis-, cancer-, and bladder function-related genes. The relevance of these gene-based findings was verified through microscopic examination of egg-exposed bladders. Our data will improve our comprehension of urogenital schistosomiasis, and may help identify new targets for diagnosis and treatment of this disease, and possibly bladder cancer and bladder-based inflammatory disorders as well.

Schistosomiasis affects up to 300 million people in regions of South America, Southeast Asia, the Middle East, Mediterranean Europe, and sub-Saharan Africa. Chronic consequences of schistosomiasis include anemia, physical and cognitive retardation in children, and organ failure. With predilection for the genitourinary tract, Schistosoma haematobium increases the risk of bladder cancer and HIV infection in women. Microscopic identification and enumeration of parasite eggs in urine (S. haematobium) or stool (Schistosoma mansoni and Schistosoma japonicum) has remained the standard for schistosomiasis diagnosis [1]. Limitations of microscopy include the need for skilled personnel in the field with microscopy equipment and its time-consuming nature. Particularly in infrastructure-limited regions, point-of-care (POC) molecular diagnostics hold the potential to transform the management of infectious diseases such as schistosomiasis that carry significant long-term morbidity if left undiagnosed. POC diagnosis could allow selective drug administration to individuals with confirmed infection rather than entire schools or communities, and an integrated diagnosis–single dose therapy approach could reduce costs of drug administration campaigns and minimize treatment-associated adverse effects. Electrochemical biosensors are well suited for molecular diagnostics because of their high sensitivity, low cost, ease of integration into POC devices, and portability of the reader instrumentation [2]. We have developed a strategy for rapid (one hour) molecular diagnosis of bacterial urinary tract infections using electrochemical biosensors [3,4]. Urinary cells are lysed and directly applied to an array of sensors functionalized with oligonucleotide probes targeting the 16S ribosomal RNA (rRNA) of common uropathogens [3,4]. Formation of the sequence-specific hybridization complex between the pathogen rRNA and the labeled capture and detector probe pairs is detected by an enzyme tag that mediates an amperometric signal output (Fig 1). Fig 1 Biosensor-based molecular detection of urinary pathogen. In this work, we demonstrate the use of our biosensor-based platform for detection of S. haematobium in urine. We developed capture and detector probes targeting S. haematobium rRNA and integrated them into our established molecular diagnostics strategy. After determining an efficient egg lysis strategy, we demonstrated direct electrochemical detection of S. haematobium eggs spiked in human urine.

Schistosoma haematobium is the etiologic agent for urogenital schistosomiasis, a major source of morbidity and mortality for more than 112 million people worldwide. Infection with S. haematobium results in a variety of immunopathologic sequelae caused by parasite oviposition within the urinary tract, which drives inflammation, hematuria, fibrosis, bladder dysfunction, and increased susceptibility to urothelial carcinoma. While humans readily develop urogenital schistosomiasis, the lack of an experimentally-tractable model has greatly impaired our understanding of the mechanisms that underlie this important disease. We have developed an improved mouse model of S. haematobium urinary tract infection that recapitulates several aspects of human urogenital schistosomiasis. Following microinjection of purified S. haematobium eggs into the bladder wall, mice consistently develop macrophage-rich granulomata that persist for at least 3 months and pass eggs in their urine. Importantly, egg-injected mice also develop urinary tract fibrosis, bladder dysfunction, and various urothelial changes morphologically reminiscent of human urogenital schistosomiasis. As expected, S. haematobium egg-induced immune responses in the immediate microenvironment, draining lymph nodes, and systemic circulation are associated with a Type 2-dominant inflammatory response, characterized by high levels of interleukin-4, eosinophils, and IgE. Taken together, our novel mouse model may help facilitate a better understanding of the unique pathophysiological mechanisms of epithelial dysfunction, tissue fibrosis, and oncogenesis associated with urogenital schistosomiasis., Author Summary Urogenital schistosomiasis (infection with parasitic Schistosoma haematobium worms, the most common human-specific Schistosoma species globally) affects over 112 million people worldwide. S. haematobium worms primarily lay eggs in the bladder, upper urinary and genital tracts, and the host immune response to these eggs is considered to cause almost all associated disease in these organs. Resulting conditions include hematuria (bloody urine), urinary frequency, fibrosis (internal scarring) of the urinary tract, increased risk of bladder cancer, and enhanced susceptibility to contracting HIV. Approximately 150,000 people die annually from S. haematobium-induced obstructive kidney failure alone, making this species one of the deadliest worms worldwide. Despite the importance of S. haematobium, a lack of an experimentally manipulable model has contributed to the paucity of research focusing on this parasite. We have circumvented the barriers to natural S. haematobium oviposition in the mouse bladder by directly microinjecting parasite eggs into the bladder wall. This triggers inflammation, hematuria, urinary frequency, fibrosis, egg shedding, and epithelial changes that are similar to that seen in clinical S. haematobium infections. Our model may provide new opportunities to better understand the basic molecular and cellular immunology of urogenital schistosomiasis and thereby contribute to the development of new diagnostics and therapeutics.