1. Background
Echinococcosis (hydatid disease) is a serious parasitic disease caused by the infection of the larvae of Echinococcosis tapeworm. The main parasitic sites of echinococcosis are liver, also in lung, brain, bone and other tissues.
There are mainly two types of the disease spread in the world, cystic echinococcosis (CE) and alveolar echinococcosis(AE). CE, also known as hydatid disease, is the most common form all over the world. CE is transmitted by dogs, and once an individual is infected by E. granulosus, the larvae of the tapeworm develop into fluid-filled cysts in various organs . As cysts grow larger over a period of time, symptoms caused by compression will arise, and permanent damage to affected organs will occur.
Fig1.life cycle of Echinococcus granulosus (sensu lato),in which humans are accidental intermediate hosts.
In China hydatid diseases mainly prevail in the pastoral areas and semi-pastoral areas of northwest provinces.The average prevalence in endemic areas was 1.08%.The hydatid diseases epidemic areas are mainly in Xinjiang, Tibet, Qinghai and other western provinces, whose economic development is relatively backward and the level of epidemic prevention is relatively poor.
Fig2.The spatial distribution of human CE in China in 2018.
In areas where Echinococcus granulosus is endemic, cystic masses in people with a history of contact with sheepdogs indicate the diagnosis of cystic echinococcosis. Imaging techniques such as CT scan, ultrasonography and MRIs are used to detect cystic lesions. After the cysts are detected, serological tests can be used to confirm the diagnosis.
In the past, surgery was the only treatment for cystic echinococcosis. Chemotherapy, cystocentesis and PAIR (percutaneous aspiration, injection of chemicals and reaspiration) have been used to replace surgery as an effective treatment for cystic echinococcosis. However, surgery is still the most effective treatment for removing cysts and can be completely cured. Some cysts do not cause any symptoms and are inactive; these cysts usually disappear without any treatment.
2. Aim
• Use RPA and CRISPR-csa12a to amplify and detect the genome of echinococcosis.
When the cyst of echinococcosis ruptures, it releases its DNA fragment, called cell free DNA (cfDNA), in human plasma. We hope to extract the cfDNA from a patient's blood and use RPA method to amplify the cfDNA of echinococcosis, and then use CRISPR-cas12a system to detect.
• Optimizing the diagnostic method of echinoccosis.
We hope to turn our project into a miniaturized kit to optimize the diagnosis of echinococcosis. RPA amplification can be carried out at room temperature, and the only material needed is a tube for the reaction system. Compared with the traditional testing methods, our project greatly reduces the requirements for equipment, but also reduces the requirements for the professionalism of personnel. Our products can be open to the public, and users do not need to have a professional medical background, which makes it have the potential for full staff testing, so we think this is the optimization of diagnostic methods.
• Reduce the prevalence of echinococcosis in epidemic areas.
The above two goals are only the phased goals that we hope to achieve, and our ultimate goal is to reduce the prevalence of echinococcosis in epidemic areas by strengthening the diagnosis and detection of echinococcosis. At present, most of the echinococcosis that can be diagnosed are in the late stage of the disease, and the treatment at this stage is very difficult. If it can be detected at an early stage, there will be a better way to treat it, and the pain of the patients will also be reduced.
3. Design
Fig3.Project flow chart of SNIPER E
In order to solve the difficulty of echinococcosis detection in hydatid disease epidemic areas, we designed this year's project-Specific New diagnostic Implement Pointing at Echinococcosis(SNIPER E), which means we will target the DNA of echinococcosis.
As mentioned earlier, hydatid disease areas are mostly in remote areas and the economic level is relatively backward.The tranditional PCR amplification technology requires specialized equipment and takes a long time. To solve this problem, we consulted the literature and some professors and decided to adopt the Recombinase Polymerase Amplification (RPA) method, which doesn't need any equipment, and the reaction time is much shorter than PCR.
CRISPR-csa12a system is a relatively mature DNA detection system, which can detect the DNA fragments we want according to the designed sgRNA, and has been widely used in our laboratory. We imagine that cfDNA is extracted from the patient's blood and then amplified with RPA. The amplified DNA was detected by colloidal gold strip after passing the CRISPR-CAS12A system. If the patient is not infected with echinococcosis, only C-band will appear on the test strip; if the patient is infected with echinococcosis, both C-band and T-band will appear on the test strip.
At present, our design has been verified in the laboratory, and we can successfully detect hydatid cfDNA in patient samples.
4. References
[1]Wan Z, Peng X, Ma L, Tian Q, Wu S, Li J, Ling J, Lv W, Ding B, Tan J, Zhang Z. Targeted Sequencing of Genomic Repeat Regions Detects Circulating Cell-free Echinococcus DNA. PLoS Negl Trop Dis. 2020 Mar 10;14(3):e0008147. doi: 10.1371/journal.pntd.0008147. PMID: 32155159; PMCID: PMC7083330.
[2]Wang LY, Qin M, Liu ZH, Wu WP, Xiao N, Zhou XN, Manguin S, Gavotte L, Frutos R. Prevalence and spatial distribution characteristics of human echinococcosis in China. PLoS Negl Trop Dis. 2021 Dec 28;15(12):e0009996. doi: 10.1371/journal.pntd.0009996. PMID: 34962928; PMCID: PMC8789093.
[3]Parasites-Echinococcosis. CDC website
