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Development of an affordable, automated and field deployable, point of care and contained system for rapid diagnosis of TB caused by Mycobacterium tuberculosis

Primary Information

Domain

Healthcare

Project No.

5400

Sanction and Project Initiation

Sanction No: 41-2/2015-T.S.I (PT.IVOL.III, ANNE-1 S.23)

Sanction Date: 09/01/2017

Project Initiation date:01/03/2017

Project Duration: 24

Partner Ministry/Agency/Industry

Ministry of Health and Family Welfare

 

Role of partner:Project investigators: Professor Dr. Seyed Ehtesham Hasnain, Kusuma School of Biological Science, Indian Institute of Technology Delhi & Vice Chancellor, JamiaHamdard University, Delhi, Dr.Ravikrishnan Elangovan, Department of Biochemical Engg and Biotechnology, Indian Institute of Technology, Delhi, Dr.Nasreen Z Ehtesham, National Institute of Pathology, ICMR Institute, Safdarjang Hospital Campus, New Delhi & Valetude Primus Healthcare Pvt (Industry Partner) led the team, conceptualized the idea, contributed in conducting the study and made all resources available for successful implementation of the project.

 

Support from partner:Ministry of health has not interacted with the team and has not contributed in giving feedback on the progress and expectations.

Principal Investigator

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SEYED E. HASNAIN
Kusuma School of Biological Science, Indian Institute of Technology, Delhi

Host Institute

Co-PIs

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Ravikrishnan Elangovan
Department of Biochemical Engg and Biotechnology, Indian Institute of Technology, Delhi

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NASREEN Z. EHTESHAM
Senior Deputy Director, National Institute of Pathology, Safdarjung Hospital.

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Prof Vijay K Chaudhary
Department of Biochemistry, University of Delhi South campus, New Delhi

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Professor Surendra K Sharma
Department of Medicine , All India Institute of Medical Sciences, New Delhi

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Dr Amita Gupta
Department of Biochemistry University of Delhi South campus, New Delhi

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Saurabh Singh
CEO, Valetude Primus Healthcare Pvt Ltd

 

Scope and Objectives

1: Development of a capture bottle with preloaded reagents for sputum thinning and sterilization.
2. Development of an automated immuno-magnetic cell enrichment system to isolate MTB cells from the sputum sample using 19 kDa surface antigen.
3. Integration of iMC2 with inbuilt fluorescent detector. Enriched MTB cells will be detected using fluorescent probe (against MTB enzymes or DNA).
4. Scalable production of reagents, capture chip and iMC2 TB device.
5. Development of POC-lateral flow based detection of MTB-specific antigens (Plan B).
6. Clinical validation on 200 TB suspects/patients.

Deliverables

We have developed a novel immunomagnetic cell capture technology that enables Mycobacterium tuberculosis (MTB) cells separation from complex sample like sputum and concentrates the cells by 100x. MTB cells enrichment by 100x without background mucus debris improved the detection sensitivity of the assay. Device and assay components are designed with scalability in mind i.e. most components of device, including single use capture chip have beenmanufactured using injection molding. All the reagents have been prepared to be stable in room temperature and assay automated to minimize human intervention. Using 100 nanometer multi-core super paramagnetic dextran coated nanoparticles, we are able to increase the efficiency of binding and using unique collection technology we are able to enrich the cells by 100x to a final volume of 10 microlitre with more than 80 percent efficiency. iMC2 TB has compact fluorimeter inbuilt in the device, the whole assay automated with exception addition of reagents in two-step. The portability and cost effectiveness of the iMC2 system allows it to deploy in all clinical settings ranging from rural to highly equipped urban labs.

 

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Scientific Output

Sputum thinning reagent (CLR) A formulation has been developed that can efficiently digest and liquefy sputum specimen thus highly simplifying the release and capture of trapped bacilli. This formulation, that we have termed as Complete Liquefaction Reagent (CLR) is a combination of various mucolytic that can efficiently digest various constituents of a sputum specimen and has now been successfully tested in the thinning of several highly purulent to non-purulent sputum specimen types. Development of a capture bottle with preloaded reagents for sputum thinning and sterilization iMC2 system has two components (Figure. 1), (A) iMC2 automated device and (B) capture chip. The system allows the enrichment or concentration of target biomarker from large volume bio-samples (e.g. Blood, feces, urine or sputum) to a small volume that in turn leads to increment in the sensitivity of the existing protocol of diagnosis. We have finalized the design and manufactured the alpha version of the device and capture bottle. These have been used in lab to test the reagents for the TB assay. Immuno-magnetic enrichment The immuno-magnetic cell enrichment system was prepared by conjugating magnetic nanoparticle (MNPs) with antibodies specific for mycobacterial surface antigen. This systemwas assessed for the capture of mycobacterial cells from liquefied sputum samples. Theimmuno-magnetic cell enrichment system has shown 80 percent capture efficiency on sputum samples (Figure. 2). Fluorescent detector device and fluorescent assay chemistry The in-house fluorescence detector has been developed and optimized to work with Fluorescein based dyes, with excitationaround 490 nm and emission around 515 nm wavelengths. Light emitting diode at 490 nm wavelength is being incorporated as the source, to illuminate the 450 microlitre of the sample and theexcited light is collected through an emission filter which is directed towards optical sensor. Theoptical sensor is a photodiode from Texas instruments which have the required electroniccircuitry with it. Presently, the fluorescence signal is being detected in electronic voltages by using a digital multi-meter.The experiments have been conducted with ten different concentrations of fluorescein dye.The steep increase in fluorescence signals was observed above 10 nM concentration of fluorescein dye (Figure. 3). Design and development of SeeTB device Along with the Imprint project, we have developed another technology called SeeTB. The SeeTB system is a compact and cost-effective way of fluorescence observation of sputum smear. The SeeTB can easily be used with different wavelength excitation based on sample staining. Thisis a novel (patent in process) planar waveguide-based illumination technology to observe fluorescence sample using bright-field microscope without any hassle of optical filters, lens installation, expensive instrumentation and without additional infrastructure upgrade. Clinical validation of performance Clinical trials for iMC2-TB and SeeTB havebeen done in Jamia Hamdard and Safdarjung Hospital. The iMC2-TB technology is being bench marked along with Gene-Xpert & culture test. We have completed the clinical validation of iMC2-TB technology on 150 sputum samples while clinical validation of SeeTB technology was done on 237 sputum samples.

 

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Results and outcome till date

iMC2 TB technology: iMC2 TB assay is a one-step diagnostic product with minimum human intervention for pulmonary tuberculosis in all kinds of healthcare settings, including the remote primary healthcare centers of the country. This system does not require any existing lab setup to process and analyze the sample. Sputum sample of the suspected patient can be collected on-site (point-of -care) directly in the capture bottle (part of test kit). Capture bottle is prefilled with sputum processing and decontamination reagents. MTB cells will be enriched to a final volume of 10 microlitre using iMC2 device followed by collection of fluorescence signal using in-built fluorescence detector. This whole process is performed in the enclosed capture bottle to avoid any exposure to health-worker and completed within 60 minutes. Device is designed to run with minimum power, with rechargeable battery to run off the grid if required and network connected. User interface and operation of test will be simplified to enable healthcare personnel trained in for basic errands in healthcare centers can use the device. See TB technology: We have developed a portable set-up called SeeTB that can convert a bright-field microscope into fluorescence microscope with minimal interventions. SeeTB, a total internal reflection based fluorescence excitation system allows visualization of auramine-O stained bacilli efficiently with high signal-to-noise ratio. We have compared the performance of SeeTB system, comprise of SeeTB set-up with thinning reagent, in 237 clinical sputum samples along with fluorescence microscope (FM), GeneXpert and liquid culture. In comparison with culture as gold standard, FM has sensitivity of 63.77 percent and SeeTB system has improved sensitivity to 76.06 percent. In comparison with GeneXpert, FM has sensitivity of 73.91 percent while SeeTB has improved sensitivity to 85.51 percent. However, there is no significant change in the specificity between FM and SeeTB system. The data demonstrates that SeeTB system provides a valid alternative to traditional FM. Unique advantages of the SeeTB system are its sensitivity, high negative predictive value, affordability, deployability and scalability. In short, the SeeTB system offers and alternative option for improved TB case identification in resource-limited settings. Moreover, large-scale field trials are required to assess the advantages and feasibility of replacing traditional FM with SeeTB as a first-line diagnostic test.

 

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Societal benefit and impact anticipated

The iMC2 system is a rapid and deployable TB diagnostic systems (Fastest phenotypic identification method) based on immuno-magnetic separation & fluorescent detection. With the contained processing cassette, the miniaturized detection is integrated into a portable device, which is promising for detection of TB at the POC. The technologies require no specialized trained healthcare technician, no sample exposure to health worker and the process is BSL3 independent. Both the devices are technologically easy to use portable device, quick for POC testing and cost effective.These devices would benefit by reducing the turnaround time for rapid results at the POC.While consideringthe clinical benefits, such as immediate treatment initiation to reduce transmission or the ability to pre-screen patients prior to more specific testing, a POC test would be more useful than the other laboratory-based test.

Next steps

Large scale production of capture bottle; Production of 10 iMC2 device; Validation of performance in 200 clinical sample; Support for development SeeTB device and process for large scale validation; Intensive technology assessment at community level will be done in partnership with Valetude Primus Healthcare, ICMR and DHR.

Publications and reports

SeeTB: A novel method to improve sensitivity of existing sputum smear microscopy Vikas Pandey, Pooja Singh, Saumya Singh, Saurabh Singh, Javid S, Naresh Arora, Neha Quadir, Nasreen E, Hasnain SE, Ravikrishnan E. (Communicated in PLOS Medicine); iMC2TB: A novel automated, deployable point of care diagnostic assay for Mycobacterium tuberculosis diagnosis. Saurabh Singh, Naresh Arora, Saumya Singh, Vikas Pandey, Pooja Singh, Javid S, Neha Quadir, Nasreen E, Hasnain SE, Ravikrishnan E. (Under Preparation); Recent progress in improving MTB diagnosis sensitive, faster, cheaper and deployable. Pooja Singh, Saumya Singh, Saurabh Singh, Vikas Pandey, Javid S, Naresh Arora, Neha Quadir, Nasreen E, Hasnain SE, Ravikrishnan E. (Under Preparation); Processing complex biological matrix for infectious disease diagnosis. Surbhi Lavanya, Saumya Singh, Saurabh Singh, Naresh Arora, Vikas Pandey, Pooja Singh, Javid S, Neha Quadir, Nasreen E, Hasnain SE, Ravikrishnan E. (Under Preparation)

Patents

Title: A composition for mucus or sputum liquefaction and a method thereof. Inventors: Hasnain SE, Nasreen E, Naresh Arora, Saurabh Singh, Vikas Pandey, Pooja Singh, Javid S, Neha , Ravikrishnan E. Trademark: SeeTB, CLR (Valetude Primus). Capture bottle design patent (Valetude Primus)

Scholars and Project Staff

Mamta Rani (SRF, 1st May 2017 to 31st Oct 2017); Dr. Swati Singh (RA; 1st Nov 2017 to 31st July 2018); Dr. Pooja Singh (RA; from 15th May 2017 to till date); Dr.Saumya Singh (SRF; 1st May 2017 to till date); Mritunjay Prasad (Project admin; 1st May 2017 to till date); Lallan (Technical Assistant; 1st May 2017 to till date)

Challenges faced

Implementation of large scale and sharp deadline project is very challenging. Team has been exhausted at the end of the project. First year money flow has empowered and enabled the project to develop two products for TB diagnostic; however, the money stopped abruptly with no clear indication of future release and rationale. It is impossible to undertake these high-risk projects without even financial sustainment of the project. Money in-flow was not commensurate with the research activity.

Other information

We have hand over the knowledge know how to industrial partner, VPH who is currently acquired funds to scale up the production and undertake the DCGI regulated clearance. Gandhian Young Technological Innovation (GYTI) award, Miniaturized Fluorescence adapter for Fluorescence Sputum Smear Microscopy using bright-field microscope. Vikas Pandey, Pooja Singh, Saurabh Singh, Indian Institute of Technology Delhi. http://gyti.techpedia.in/award-list-2018.php. Valetude Primus Healthcare has received support via the Grand Challenges Exploration program. ICMR has suggested to submit a proposal for large scale validation and impact study of SeeTB.

Financial Information

  • Total sanction: Rs. 22920000

  • Amount received: Rs. 13501000

  • Amount utilised for Equipment: Rs. 4025821

  • Amount utilised for Manpower: Rs. 3964478

  • Amount utilised for Consumables: Rs. 6729969

  • Amount utilised for Contingency: Rs. 123408

  • Amount utilised for Travel: Rs. 33903

  • Amount utilised for Other Expenses: 0.00

  • Amount utilised for Overheads: Rs. 990800

Equipment and facilities

 

Refrigerators (-20 & 4 degree); Weighing balance; pH meter; OrbitekShaker; N-biotek Incubator shaker; Biosafety hood x 2; multimode fluorescence reader;fluorescence camera; fluorescence microscope; autoclave