One Day Path Catch- ODPC

In clinical scenario diagnosing infectious disease by detecting the right pathogen is very crucial. We have several existing assays in market to detect known organisms. Bacterial specific, fungal specific, viral specific and zoonotic specific targeted assays. But there are limitations for these existing assays as all has to be performed sequentially which consume time and money a lot. In this scenario, implementation of next generation sequencing can potentially assist in clinical diagnosis and we present an assay ODPC, a metagenomic approach for identification of all pathogens like bacteria, fungi, RNA /DNA virus,and other zoonotic species in a single diagnostic assay in a day time. The challenge of deciphering unknown medical cases can be solved by this assay. ODPC can come up with identification of new pathogens that are reason for a new cause. In accordance with the reads obtained for each organism, we can conclude on severity of the situation.

As of now the available kits in market used by clinicians are mainly Real Time PCR based. Sanger sequencing based identification  though  reliable fails if the infection is polymicrobial. If it is caused by single organism, the final report can be obtained in 3 days time which might delay a perfect diagnosis and intervention. Real time based kits are mainly probe based and handles only limited number of targets. Blood culture considered as gold standard byclinicians can detect the living organisms in sample but has low sensitivity as many of the organisms might be slow growing and chances of omitting intracellular pathogens are more and hence clinicians are eager to have new technologies that can identify all organisms present in the sample in a very less time interval and expect to get the diversity ratio of polymicrobes present so that effective treatment can be given to the patient. Here we are presenting an assay that can be completed in a day time with all accuracy, diversity and economically very feasible. This test can identify all type of infectious organisms like, bacteria, fungus, DNA Virus, RNA virus, fungi and parasites in a test. Sepsis can be handled well with this assay.

Simultaneously total DNA from whole blood/ throat swab/spit sample  and RNA from filtered plasma isolated undergoes a cDNA synthesis with a K-8Nmer and further amplified with K-mer. DNA is multiplexed with 16S bacterial , ITS Fungal and 18S Parasitic primers and the amplicons are mixed with viriome amplicon in equal nanomol concentration and fragmented to a standard library size so as to run in 150x2 PE I seq 100 Illumnina platform/Genapsys platform.The pre-processed reads are aligned to the host genome using bowtie aligner tool in order to remove the host reads. A fasta file is created from the final fastq files in order to perform blast search against nr database. The blast results are parsed and classified according to the organism using custom made perl script. The organism based annotation are done. Since we aim for only 50K reads per sample in this assay, minute presence of insignificant organisms which are irrelevant for infection will not be captured but a control sample healthy control will be maintained in each run so as to nullify any contamination associated with handling the sample. Sudden out breaks like Covid 19 could be handled with out misinterpretation due to variation due to evolution.

This can be useful in case of local outbreak of unknown diseases, transfusion transmitted infections, surgical site infections, haemopoietic stem cell transplantation, solid organ transplants etc. All these cases might go fatal if not diagnosed at right time. In present COVID 19 scenario, evolving variants can give false negatives. The factors that prevent the right diagnosis with existing assay and technologies is time limitation and specificity and sensitivity. Several individual methods are available which are type specific or amplification based, which cannot identify or reveal variants. Hence a new assay is awaited by clinicians to identify any pathogen at least turn around time. Several samples can be multiplexed in experiment so as to reduce the cost to an affordable limit. These factors make it unique from the existing kits and tests available in market. An error free testing can be assured with this kind of methods in outbreaks like Covid 19 is essential. Death of sepsis/local outbreaks may be  due to secondary infection and the high organism infected could be traced with the same assay and can interfere immediately with effective antibiotic to save the life.

How can communities around the world prepare for, detect emerging pandemics, taking the cases of Coronavirus disease 2019 (COVID-19) is the latest in a series of infectious disease emergencies, including cholera, Ebola, SARS, Chikungunya, HIV/AIDS, and influenza world should be ready to take up any pandemic at any time. We should be ready to diagnose the infections at right time though we are unaware of source. The test developed can handle any unknown cases and have 100% sensitivity being genome based confirmation and depth of infection available with read depth. This is how this innovation aligns with challenge called for.

Existing technologies depend on RealTime PCR , that too possible only for known organisms. NGS tests are available from Thermo, illumina etc which are all panels which are probe based or primer based. So chances of false negatives are there if it has variants. Here we are separating the viral particles and sequencing completely, so that we won't make unnecessary data of host genome. We are multiplexing bacterial, fungal and zoonotic parasites and make a single library so to reduce the cost to minimum and sequence and annotate the sequence so as to find the infectious organism and depth of the infection. In cases of sepsis, local out breaks and pandemics we  won't get time to check each and every susceptible organisms and hence a test of this kind is required.

NGS is a widely used technology. 

Gwinn M, MacCannell D, Armstrong GL. Next-Generation Sequencing of Infectious Pathogens. JAMA.2019;321(9):893–894. doi:10.1001/jama.2018.21669

But our process is innovative and economically feasible.

We should utilize more reliable and most modern technology for reliable results. We should utilize the resources kept idle at the time of pandemics if it helps  for the wellbeing of human kind.

We are in pilot stage, We have done it at research level only, we need to take it up for a service level. 

In 2020, we want it to be validated with some pandemic samples and get in to market. In five years our plan is to establish service in different countries.

We need to buy an NGS equipment, being start up and self funded , now it seems to be not practical. Technically we need to validate for pandemics like COVID 19. 

We are trying for government funding. After procuring the instrument we will be validating the pandemic samples.

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1. PI  -1 (Full time)

2. Consultant Bioinformatician-1  (Part time)

3. Advisor - Virologist-1 (Part time)

4. Research Associates Lab-2  (Full time)

5. Bioinformatician -1 (Full time)

• Dr. Beena P.S. holds a Ph.D in Biotechnology from Cochin University of Science and Technology. Her expertise includes Genomics, Proteomics, Molecular Biology and Bioprocess technology. She has a total of 18 years of experience in Genomics and proteomics that includes 10 years of industrial experience in Genomics Service Industry and several years of research experience in academic research with exposure to project management, ICH GCP guidelines and biosafety measures.
• Team Members: 
• 1. Ms. Annu George- MSc Bioinformatics with `2 years experience in Bioinformatic analysis
• 2. Mr Jebin James- MSc Biotechnology with 1 Year experience in molecular biology Techniques
• 3. Ms. Athira KR-    MSc Biotechnology with 1 Year experience in molecular biology Techniques   
•  
• Consultant: Dr. Bipin Balan- PhD in  Bioinformatics from SCIENZE AGRARIE, ALIMENTARI,  FORESTALI E AMBIENTALI, University of Palermo, Italy. 

& Visiting Ph.D Student – Department of Plant Science, University of California, Davis, CA, USA.) More than 10 years of experience in developing pipelines and highthrough put sequencing data analysis.Several peer reviewed publications to his credit.

Advisor: Dr. Veena P. Menon - PhD from   Dept. of Microbiology & Immunology, Georgetown University, Washington DC, USA 

Accomplished Biomedical Scientist, with 20 years of experience,  with a focus on the pathogenesis and immune response to infections. 

 Demonstrated expertise in planning/setting up a BSL2/BSL3 level clinical virology laboratory. Technical lead in the development of rapid and new multiplex diagnostic assays as well as platforms in the hospital for infectious diseases. 

 An excellent published track record in virology, immunology and microbial genetics. 

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B to C and B to B

B to C, we can provide directly to patients.

Bto B - We can provide to hospitals, Government Health care agencies at the time of requirements.

We are providing several genomics and proteomics services for a constant cashflow.  We are looking for grants to establish the above said service.