Worm RNA from India
Flying our ideas to reality – Two team-members travelled to India to learn about the environment our test needs to function in and to build up a network that helped us being the first to decode the transcriptome of lysed T. solium eggs and to prepare a clinical study for our diagnostic test.
T. solium is endemic in India
Data from the WHO indicate that infections with the pork tapeworm is endemic in India. When performing a literature review, we found only few small studies examining the prevalence of T. solium in India. So we were full of uncertainty, when we sent two team members to India in order to explore the context in which our diagnostic test might be used in the future.
Meeting the People's Vigilance Committee on Human Rights
Upon our arrival, we met Dr. Lenin from the Human Rights organization PVCHR, who gave us the chance to accompany social workers to Musahar communities, where poor and discriminated people live in small collections of huts. PVCHR suspected a high chance that people in these villages are infected with the pork tapeworm.
Standard of T. solium diagnosis in India
At this point we learnt how important it is to develop a rapid field test: Currently the infection is diagnosed by microscopy, but trained stuff and equipment are usually located in regional hospitals close to the hospitals. Driving to the next hospital can take more than two hours. Unfortunately, the villagers are forced to work long hours and in harvesting season even 6-7 days a week to secure a minuscule income for their families. Many of them never went to a hospital and are superstitious. Therefore, we and PVCHR believe it would be best, if trusted social workers handle our test on site in the village.
Next, we went to a government hospital, a private hospital and several clinical laboratories to meet physicians and to discuss our idea and their thoughts on T. solium. Many physicians, in particular Dr. Dilip Mishra (picture), were very interested in a rapid diagnostic test and we agreed to a joint research project. While we were still on site, we visited the research facilities and planned how to isolate RNA from T. solium in this environment. Our plan was to disrupt the eggs using the highly corrosive detergent “QIAzol” and a homogenizer.
Subsequently, we planned to ship the lysate containing the worm’s RNA in QIAzol to Germany for further purification and analysis. However, we realized the difficulties to export a shipment containing biological compounds and a phenolic agent. For this reason, we traveled to Delhi to meet iGEM IIT Delhi. Together with the Delhi-Team, we established an RNA isolation pipeline. This posed to be a real challenge, because regular RNA isolation kits are not developed for temperatures above 40°C. After successful isolation, the RNA is dissolved in water and for this reason easy to ship.
After our return to Germany, Dr. Mishra obtained intact eggs from Taenia Solium. With the equipment and technical advice, he created RNA-Lysates from these eggs for the first time. Unfortunately, the export of the lysates stabilized with QIAzol took three months due to difficulties to acquire the needed certificates. Thanks to the help of iGEM IIT Delhi, we were ultimately able to ship the samples to Germany, to extract the RNA from the Lysates. Finally, the sequencing core facility of the Max-Planck-Institute for Molecular Genetics, Berlin, generated two libraries from the RNA and to sequenced it using Next-Generetion-Sequencing on a HiSeq5000. Sequencing yielded 96 million reads that characterize a unique sample of T. solium eggs. To our knowledge, we are the first to report a method direct RNA isolation from T. solium eggs without a manipulative hatching procedure and to perform transcriptome analysis thereof. This information is crucial for the development of diagnostic tests.
Simultaneously, we wrote an application to the institutional review boards to gain ethical consent for a clinical study. We received ethical consent from both boards. To our knowledge, we are the first iGEM team to write an application and received the ethical approval for a clinical study. Throughout this study, we will accompany social workers, that provide tapeworm-chemoprophylaxis to villages and recruit volunteers for a stool sample prior to receiving chemoprophylactic treatment. (as part of a preventive program supported by the Indian Ministry of Health). The sample will be analyzed by Dr. Mishra using light microscopy as the current standard for diagnosis of T. solium in India. The remaining sample is used to test a prototype of our diagnostic test platform. First, we will evaluate, whether our sensor system will be a suitable option for the rapid diagnosis of T. solium within an Indian diagnostic laboratory. Secondly, we plan to gather more material of tapeworms to generate molecular data for basic research or eventually, refinement of our own test. Next to validating our test, this study aims to evaluate the epidemiology of tapeworm infections in India. Once completed, it will be the third largest clinical study examining the prevalence of T.solium in India.
Our Approach to NTDs
The aim of our project, facilitating diagnosis of T. solium infection, is a topic set within the field of synthetic biology, but with connections to many other domains. The targeted infection is listed as one of the 20 communicable diseases that are mostly affecting poor populations – a neglected tropical disease (NTDs). Stopping the spread of such diseases is thus not only a matter of medical progress, but also of an improved education, higher hygiene and sanitation standards as well as of more equitable communities. This also means that the challenge of NTDs is not only taken on by biologists and physicians! Politicians, health workers and NGOs as well as the private sector have for a long time made efforts to eliminate these diseases. With the NTD lab, our team aimed to bring all these players together to discuss how innovation in the field of these poverty-associated diseases could look like.
On the 17th of May 2017, we thus invited more than 100 participants to the premises of the Humboldt Graduate School. The event was split into two main parts: in an interactive “project fair”, our team presented its work to the participants next to projects of NGOs such as the Christoffel Blindenmission (CBM) or of other scientific institutes such as HTW Berlin. After a reception, a panel discussion with eminent speakers ensued. We were happy to welcome Dr. Georg Kippels (member of the parliament), Prof. Dr. KH Martin Kollmann (member of the advisory board of DNTDs and scientific advisor to the CBM Kenya), Dr. Joachim Klein (German Ministry for Research and Education), Dr. Maria-Luisa Rodriguez (Global Program Head Nifurtimox, Bayer AG) as well as our team leader Henrik on the podium. In an insightful discussion, we were discussing not only the scientific requirements for tackling the problem of NTDs but even how science might be a driving force in reaching the sustainable development goals.
Organizing an event involving almost 150 people was no small endeavor to our team – so what was the goal we aimed to reach with the symposium? Early in our project we realized that neglected tropical disease not only warranted our attention, but needed more general awareness. A broader public should know about these diseases to make a joint action against NTDs possible. We are happy to report that we reached our goals in two different ways: On the one hand, the many students in the audience very visibly curious to find out more about these poorly understood diseases and we even gained new team members. On the other hand, our invited speakers were impressed at the many questions and informed statements from the audience and realized that the topic of NTDs is also interesting to non-experts. Our partners, NGOs as well as scientific groups, expressed interest in organizing a second NTD lab next year – our plans on making this event involve an even larger audience were favorably met, and the planning phase has already started!