Periodic Reporting for period 1 - FIT-UTI (Fully Integrated Technology based on quantitative polymerase chain reaction for the diagnosis of Urinal Tract Infection)
Periodo di rendicontazione: 2017-09-01 al 2019-08-31
Urinary tract infection (UTI) is a symptomatic bacterial infection that occurs within the urinary tract and is one of the most prevailing infectious diseases with a substantial financial burden on society. It is indeed estimated that 15% of all community-prescribed antibiotics in some EU countries are dispensed for UTI1. With the alarming state of microbial resistance development, it is vital to develop fast point of care tools to detect UTI as well as to record its resistance to medical treatments. Yet, the diagnosis of UTI by specialists/doctors in hospitals or clinics is not an easy task due to the small timeframe of visits, thus there is an urgent need of point-of-care devices that give fast and reliable diagnosis within a comfortable waiting time (<30 mins).
Urine dip stick test is the basic and most frequently used method for UTI diagnostic testing. Such tests can detect nitrite, pH, leukocyte esterase, protein, glucose, etc. The result is constant, however, urine dip sticks suffer from a relatively low sensitivity and specificity. Only if elevated leukocyte esterase that points to the presence of white blood cells is tested, further bacteriological urine culture, which is the gold standard for a UTI test, will be performed to identify the specific pathogens under microscopy. Despite its acceptance, this technique is still suffering from various shortcomings: it is laborious, time-consuming and expensive since it requires specialized laboratory facilities to perform each test.
To overcome the shortcomings of existing diagnosis tools, this project aims at developing a new diagnosis tool using genetic identification based on quantitative polymerase chain reaction (qPCR) methods for UTI diagnosis. qPCR is a rapid method for quantification of nucleic acid sequences that combines PCR chemistry with fluorescent probe detection of amplified DNA segments. For point-of-care applications, miniaturization of PCR devices leads to faster DNA amplification process and lower costs of the biological sample consumption. Several integrated microfluidic nucleic acid testing devices have been reported, however, to facilitate tests at the point of care, it is ideal to integrate biosample preparation with the PCR reactions and fluorescence analysis into a single platform. For UTI diagnostics, it means that DNA of the infectious bacteria (e.g. Escherichia coli) need to be purified from patient urine and specific gene target is investigated afterwards. Such reported point-of-care diagnostic devices are rare because of the following challenges: (i) to perform cell concentration and lysis full-automatically; (ii) to integrate sample preparation and analysis into a single system; (iii) to miniaturize the plug-and-play operating system; (iv) to reduce the cost of the disposable chip.
This project is based on BforCure proprietary FASTGENE technology, the world fastest qPCR technique which has already been tested on Anthrax and Ebola simulating agents. Within this project, a fully automatic lab-on-a-chip cell concentration system has been developed first. Then we validated the workflow from our developed cell-concentration system to PCR analysis. This automatic system has been interested by a couple of industrial partners and we successfully did a proof-of-concept demo to them, such as legionella detection in big volume (1L) tap water. At the meantime, we developed a prototype integrating urine sample preparation and qPCR for bacteria gene recognition for point-of-care UTI diagnostics. The prototype is aimed to perform bacteria filtration, lysis, PCR reagents mixing and PCR full-automatically within 30 minutes. It is also can be extended to many other applications, such as defense and security, water treatment, food safety monitoring, etc.
1 Guidelines on urological infections, European Association of Urology, 2015
Urine dip stick test is the basic and most frequently used method for UTI diagnostic testing. Such tests can detect nitrite, pH, leukocyte esterase, protein, glucose, etc. The result is constant, however, urine dip sticks suffer from a relatively low sensitivity and specificity. Only if elevated leukocyte esterase that points to the presence of white blood cells is tested, further bacteriological urine culture, which is the gold standard for a UTI test, will be performed to identify the specific pathogens under microscopy. Despite its acceptance, this technique is still suffering from various shortcomings: it is laborious, time-consuming and expensive since it requires specialized laboratory facilities to perform each test.
To overcome the shortcomings of existing diagnosis tools, this project aims at developing a new diagnosis tool using genetic identification based on quantitative polymerase chain reaction (qPCR) methods for UTI diagnosis. qPCR is a rapid method for quantification of nucleic acid sequences that combines PCR chemistry with fluorescent probe detection of amplified DNA segments. For point-of-care applications, miniaturization of PCR devices leads to faster DNA amplification process and lower costs of the biological sample consumption. Several integrated microfluidic nucleic acid testing devices have been reported, however, to facilitate tests at the point of care, it is ideal to integrate biosample preparation with the PCR reactions and fluorescence analysis into a single platform. For UTI diagnostics, it means that DNA of the infectious bacteria (e.g. Escherichia coli) need to be purified from patient urine and specific gene target is investigated afterwards. Such reported point-of-care diagnostic devices are rare because of the following challenges: (i) to perform cell concentration and lysis full-automatically; (ii) to integrate sample preparation and analysis into a single system; (iii) to miniaturize the plug-and-play operating system; (iv) to reduce the cost of the disposable chip.
This project is based on BforCure proprietary FASTGENE technology, the world fastest qPCR technique which has already been tested on Anthrax and Ebola simulating agents. Within this project, a fully automatic lab-on-a-chip cell concentration system has been developed first. Then we validated the workflow from our developed cell-concentration system to PCR analysis. This automatic system has been interested by a couple of industrial partners and we successfully did a proof-of-concept demo to them, such as legionella detection in big volume (1L) tap water. At the meantime, we developed a prototype integrating urine sample preparation and qPCR for bacteria gene recognition for point-of-care UTI diagnostics. The prototype is aimed to perform bacteria filtration, lysis, PCR reagents mixing and PCR full-automatically within 30 minutes. It is also can be extended to many other applications, such as defense and security, water treatment, food safety monitoring, etc.
1 Guidelines on urological infections, European Association of Urology, 2015
The work performed during this project can be mainly divided into three parts, the first part is to develop the pre-treatment microfluidic device to concentrate cells; the second part is the bacteria (E. Coli) test to find out the limit of the detection; the third part is to further optimize the FASTGENE qPCR analysis part.
Generally, for the pre-treatment part, we managed to concentrate > 100 bacteria suspended in 10 ml water to 150 µl nuclear-free water fully-automatically in less than 1 minutes. The yield efficiency is more than 50%. About the bacteria test results, we confirmed the limit of detection can reach as less as 100 bacteria. For the qPCR analysis part, since we are aiming for developing a product, we did lots of optimization on it, e.g. thermal heating controlling, optical sensors adjusting, software improvement. The result is that the we can amplify the specific gene of E.Coli in BforCure self-developed qPCR machine 240 times more in 11.3 minutes and get the quantitative result is real time. In the end, we designed a new prototype to integrate these steps in sequence and at the same time try to miniaturize it to make it portable.
The result of the qPCR module developed in this project will be the first product in BforCure. The product name is CHRONOSTM. Its market will be focusing on R&D labs. I studied almost all the competitors worldwide and defined the product features. We made the product design and it is announced on BforCure website (www.bforcure.com). I analyzed the manufacturing cost and defined the market price. I did a mailing campaign (20,000 potential contacts) to advertise our product and got quite positive feedback. More than 40 customers from worldwide are eagerly waiting for our on-site demo. Now the product development is under the final procedure to reduce the cost and it will be officially launched on the market at the beginning of 2020.
The more advanced version integrating sample preparation and qPCR will be the second product in BforCure. Besides its application in UTI diagnosis, it has attracted interests of industrial partners as well, such as in defense and security, water treatment and fuel contamination detection and we already signed the collaboration contracts with partners in each field to co-develop this product.
The ER participated a couple of congress and international conferences with the marketing & sales team of Elvesys, e.g. 2017 MEDICA, 2018 ICMFLOC, 2018 µTAS, to meet and discuss with the potential customers. She participate to one workshop.
Generally, for the pre-treatment part, we managed to concentrate > 100 bacteria suspended in 10 ml water to 150 µl nuclear-free water fully-automatically in less than 1 minutes. The yield efficiency is more than 50%. About the bacteria test results, we confirmed the limit of detection can reach as less as 100 bacteria. For the qPCR analysis part, since we are aiming for developing a product, we did lots of optimization on it, e.g. thermal heating controlling, optical sensors adjusting, software improvement. The result is that the we can amplify the specific gene of E.Coli in BforCure self-developed qPCR machine 240 times more in 11.3 minutes and get the quantitative result is real time. In the end, we designed a new prototype to integrate these steps in sequence and at the same time try to miniaturize it to make it portable.
The result of the qPCR module developed in this project will be the first product in BforCure. The product name is CHRONOSTM. Its market will be focusing on R&D labs. I studied almost all the competitors worldwide and defined the product features. We made the product design and it is announced on BforCure website (www.bforcure.com). I analyzed the manufacturing cost and defined the market price. I did a mailing campaign (20,000 potential contacts) to advertise our product and got quite positive feedback. More than 40 customers from worldwide are eagerly waiting for our on-site demo. Now the product development is under the final procedure to reduce the cost and it will be officially launched on the market at the beginning of 2020.
The more advanced version integrating sample preparation and qPCR will be the second product in BforCure. Besides its application in UTI diagnosis, it has attracted interests of industrial partners as well, such as in defense and security, water treatment and fuel contamination detection and we already signed the collaboration contracts with partners in each field to co-develop this product.
The ER participated a couple of congress and international conferences with the marketing & sales team of Elvesys, e.g. 2017 MEDICA, 2018 ICMFLOC, 2018 µTAS, to meet and discuss with the potential customers. She participate to one workshop.
Benefiting from the ultra-fast speed of BforCure’s proprietary FASTGENE technology, compared to the similar products on the market, the competitiveness of the BforCure’s products is the ultra-fast performance. By integrating with the automatic sample preparation procedure, which has been technically validated in this project, it provides an on-site diagnostic solution to prevent biological hazards by biosensing in order to reduce health impacts and unnecessary use of hazardous chemical and antibiotic treatments. The output of this project, with the further development in BforCure in the following years, will become a mature product on the UTI point-of-care diagnosis market. It will be extended into many other industrial applications, such as analyze and monitor the presence of bioterrorism agents on the field, or pathogenic microorganisms in industrial environments as well as in nature.