Team:Lambert GA/Part Collection
Project Description
Soil-transmitted helminthiasis infects 1.5 billion people globally. The prevalence and persistence of these parasitic worm infections stem from poor sanitation infrastructure and a lack of affordable diagnostic tools. LABYRINTH, a helminth detection system, implements low-cost hardware devices, biosensor toehold switches, and software analysis to diagnose helminthiasis. Using Caenorhabditis elegans as a model organism for infectious helminths, LABYRINTH isolates and lyses helminth eggs using a frugal filter and homogenizer. Biosensor cells detect C. elegans by targeting the F59G1.6 gene with an RNA toehold switch. If transcribed, the toehold switch activates GFP expression, indicating the presence of helminth eggs in the sample. The FluoroCents app quantifies fluorescence and maps this data onto a cloud-based service, enabling health organizations to efficiently allocate targeted anthelmintic medications. LABYRINTH has the potential to improve the quality of life for over a billion people worldwide by illuminating the chronic nature of helminthiasis and increasing the affordability of diagnostics.
HELMINTH BACKGROUND
Helminths, parasitic worms, are the most common infectious agent afflicting members of developing countries. They are transmitted through mediums including soil, water, and fecal matter, which makes the infection is most prevalent in developing countries that lack access to medical facilities and proper hygiene. Approximately 300 million individuals with serious helminth infections suffer from severe morbidity that results in more than 150,000 deaths annually. In addition to the immediate health consequences, helminth infections prevent educational advancement and hinder economic development. Symptoms of those infected include impaired child development, risky pregnancy, and loss of productivity.
Helminths are characterized as invertebrates with elongated bodies that can either be flat or round. There are 2 major groups of parasitic helminths: roundworms (nematoda) and flatworms (platyhelminthes). Of the many species that exist, half are parasitic. Parasitic species often have limited digestive tracts, nervous systems, and locomotor abilities. Helminths are able to survive in their hosts for many years thanks to their ability to manipulate the immune response by secreting immunomodulatory products. All parasitic worms produce eggs for reproduction. These eggs have a strong shell that protects them against a range of environmental conditions allowing them to survive outside their hosts in the environment for many months.
The 4 most common Soil Transmitted Helminths are roundworm (Ascaris lumbricoides), whipworm (Trichuris trichiura), and hookworm (Necator americanus and Ancylostoma duodenale). A recent census suggests A. lumbricoides infects 1.221 billion people, T. trichiura 795 million, and hookworms 740 million (Disease Control Priorities in Developing Countries). Of the 5.2 million people with STHs globally, 62% are caused by the hookworm parasite. The greatest number of STH infections occur in the Americas (Caribbean), East Asia, and Sub-Saharan Africa.The life cycles of Ascaris, Trichuris, and hookworm follow a general pattern Most parasitic nematodes lay eggs that contain either a zygote or a completely formed larva. Some nematodes, such as Trichinella spiralis, produce larvae that are deposited in host tissues. The developmental process involves egg, larval, and adult stages. There are 3 types of transmissions: direct, modified direct, or skin penetration. Direct infection occurs when eggs are transmitted from anus to mouth without reaching soil, such as Enterobius vermicularis (pinworm/threadworm) and Trichuris trichiura (whipworm). Modified direct infection occurs when eggs passed in feces only become infectious following incubation time in soil, which is exhibited in roundworm. Skin penetration is used by hookworms (Necator americanus). The adult parasite stages inhabit the gastrointestinal tract (Ascaris and hookworm in the small intestine; Trichuris in the colon), reproduce sexually, and produce eggs, which are passed in human feces and deposited in the external environment.
The lifetime of adult worms varies from one species to another, but it is generally in the range of one to eight years. This lifetime of several years is a result of their ability to manipulate the immune response of their hosts by secreting immunomodulatory products. The shell of a helminth egg consists of three layers: a lipoidal inner layer, a chitinous middle layer, and an outer protein layer. Hundreds of thousands of eggs are produced each time the female worm deposits its eggs - called oviposition. Larvae hatch from these eggs, either inside or outside the host, depending on the type of helminth. Life cycles of helminths differ in specific aspects. The larvae that mature in the host take from two weeks up to four months depending on the species. Eggs can reach the earth when polluted wastewater, sewage, or human waste enter the soil. The eggs are the infective stage of the helminth' life cycle for causing helminth diseases.
For trichuriasis, most patients are asymptomatic. However, heavy infestations may cause severe gastrointestinal symptoms such as colitis and dysentery. Iron deficiency anemia is common within children with poor diets. Children are easily diagnosed, but in older patients, stool microscopy and full blood counts are useful. Hookworm infections, like trichuriasis, are also asymptomatic. Patients infected with Hookworm will often experience abdominal discomfort, anorexia, nausea, vomiting and diarrhea (sometimes containing blood and mucous). “Ground itch” may occur at the site of penetration, and a rash of cutaneous larva migrans may be seen in hookworm infections. The primary treatments for hookworm are an immediate dose of albendazole (400mg) or mebendazole (100mg). The WHO recommends preventive chemotherapy in endemic areas. Anthelmintics used alone or in combination prevent death and contribute towards a sustained reduction in the endemic. Since many anthelmintics are broad-spectrum, preventive chemotherapy interventions are drug-based rather than disease-based.
Many agencies, programs, and civil societies are working to integrate STH control through targeted anthelmintic drug treatments.
Inspiration/Project Motivation
Dominican Republic
On the first week of April of 2019, members of Lambert iGEM traveled to Hato Mayor, a small community in the Dominican Republic, to install water filters. We returned days later overwhelmed by the resilience and optimism of the community, shocked by the dire conditions of their public health system, and motivated to join the fight against the world’s most neglected tropical diseases: helminth infections. During our trip, we witnessed the daily struggles of a community lacking access to proper sanitation infrastructure, from long walks for clean water to young children suffering from parasitic infections. Their stories brought us to local health professionals at the front lines of this public health battle. The consensus of the community was clear - poor sanitation conditions cause serious illnesses and impede many from attaining goals such as higher education and economic viability. The root of this issue is accessibility. Lack of affordable and effective diagnostics in third-world health systems expose millions of people to neglected tropical diseases, mainly parasitic infections. Prevention and diagnosis of such diseases are often out of reach for most citizens in these developing nations. Helminth infections in particular are treatable, but species diversity and availability deter the effectiveness of known anthelmintics. As a result, helminth infections are too common in these neglected tropical areas, a potentially fatal disease regarded as just a part of life. Driven by our experiences with the community of Hato Mayor, Lambert iGEM aims to disrupt this status quo by developing a frugal diagnostic tool for helminth infections. We believe an accessible and effective test will transform these broken health systems, allowing easy identification, diagnosis, and treatment for disadvantaged communities around the world.
Defining the Problem
Soil-transmitted helminths (STHs), a neglected tropical disease, infect 1.5 billion people (24% of the world’s population) worldwide (World Health Organization [WHO], 2019). The current method of diagnosis utilizes the ova and parasite exam, which is a microscopic examination of stool samples by an expert to search for parasites that may infect the lower digestive tract (Khurana & Sethi, 2017). For typical stool testing, fresh samples must be collected and taken to a laboratory within 2 hours, or the samples need to be maintained in specific transport vials with preservative solutions in order to get the most accurate results (LabCorp, 2019). The specificity of this test is often cost prohibitive to individuals who reside in low-income areas. The cost of one ova and parasite exam is approximately $70, excluding shipping. It takes 7-10 days for ova and parasite test results excluding any additional delays due to the weather, holidays, and lab delays. Because some parasites do not shed into the stool until they have reached a certain threshold, some labs request that multiple samples are submitted over several days in order to improve the sensitivity of the results (Walk-In-Lab, 2017). The intense labor, the associated cost, and the low sensitivity of this exam result in many individuals choosing to forgo testing.
Another issue of increasing importance is the rise of anthelmintic resistance in medicine. The most common anthelmintics are used to treat parasitic infections are albendazole and mebendazole due to their broad spectrum effectiveness (Ryan, 2018). Unfortunately, the widespread use of these anthelmintics has led to reports of increased resistance from some species of helminths, and the continued overuse of these general anthelmintics may render them useless in the near future (Castro et al., 2019). By creating a system that is able to identify specific helminths in a more efficient way, Lambert iGEM can hope to stimulate the development of more distinct anthelmintics. This targeted approach may help to treat patients according to their individual needs and eliminate reliance on general anthelmintics.
Approach
Lambert iGEM has created a new system for detecting various helminth species using hardware devices and a toehold switch mechanism specific to Caenorhabditis elegans. Rapid maturation, easy manipulation, a sequenced genome, and anatomical similarity to most nematodes make this species the ideal model organism for the proof of concept. A 3D-printed filter and frugally designed bead homogenizer is first used to isolate the helminths from the fecal sample of an infected individual and expose the DNA by breaking open the outer chitin layer. After undergoing transcription, the resulting RNA containing complementary sequences to the toehold domain binds to the hairpin stem and unravels the loop. This exposes the ribosomal binding site and start codon, allowing translation of GFP, the reporter protein, to occur. Lambert iGEM’s low-cost, portable fluorometer then quantifies the intensity of the GFP produced by the toehold construct. Through the demonstration of the C. elegans model, Lambert iGEM hopes to encourage similar applications involving specific species of pathogenic helminths. C. elegans’ anatomical similarity to most nematodes, rapid maturation, easy manipulation, and sequenced genome makes it the ideal model organism for the proof of concept.
REFERENCES
[1] Hotez, P. J., Brindley, P. J., Bethony, J. M., King, C. H., Pearce, E. J., & Jacobson, J. (2008). Helminth infections: the great neglected tropical diseases. Journal of Clinical Investigation, 118(4), 1311–1321. doi: 10.1172/jci34261
[2] What are helminths? (2015, November 17). Retrieved September 24, 2019, from https://www.yourgenome.org/facts/what-are-helminths.
[3] CDC - Parasites - About Parasites. (2016, April 22). Retrieved September 24, 2019, from https://www.cdc.gov/parasites/about.html.
[4] Cox, F. E. G. (2002, October). History of human parasitology. Retrieved September 24, 2019, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC126866/.
[5] Cox, F. E. G. (2002, October). History of human parasitology. Retrieved September 24, 2019, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC126866/.
[6] CDC - Hookworm - Biology. (2019, September 17). Retrieved September 24, 2019, from https://www.cdc.gov/parasites/hookworm/biology.html.
[7] Hedley, L., & Serafino Wani, R. L. (2015). Helminth infections: diagnosis and treatment. The Pharmaceutical Journal. doi: 10.1211/pj.2015.20069529
[8] Mirisho, R., Neizer, M. L., & Sarfo, B. (2017). Prevalence of Intestinal Helminths Infestation in Children Attending Princess Marie Louise Children’s Hospital in Accra, Ghana. Journal of Parasitology Research, 2017, 1–7. doi: 10.1155/2017/8524985
[9] Helminth Infections and their Impact on Global Public Health. (2014). Public Heath England. doi: 10.1007/978-3-7091-1782-8
[10] Pineda, M. A., & Harnett, W. (2014). Immunomodulation by Parasitic Helminths and its Therapeutic Exploitation. Immune Response to Parasitic Infections, 175–212. doi: 10.2174/9781608059850114020011
[11] Saboyá, M. I., Catalá, L., Nicholls, R. S., & Ault, S. K. (2013). Update on the Mapping of Prevalence and Intensity of Infection for Soil-Transmitted Helminth Infections in Latin America and the Caribbean: A Call for Action. PLoS Neglected Tropical Diseases, 7(9). doi: 10.1371/journal.pntd.0002419