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Six Stages to Developing a Novel Infectious Disease Assay

Human Health In Vitro Diagnostics

Advancing Diagnostic Technologies for Respiratory Illness

Respiratory illnesses are generally characterized by a fever, a cough, and a sore throat. These kinds of infections are common during fall and winter and can occur in either the upper or lower respiratory tract. As the winter season gives way to spring, you may be reflecting on your own recent bouts of illness. Within, we recommend six stages to developing a novel infectious disease assay in advance of the next respiratory virus season.  

The etiologic agents of respiratory tract infections, whether bacterial or viral, can cause various symptoms. Depending on the location of infection, this might include pharyngitis, bronchitis, and even pneumonia in some cases. Recent activity levels of respiratory illness are relatively high in the United States. Data shows that COVID-19, influenza, and RSV combined were responsible for 10 percent of ER visits during the past two winters. In fact, these three viruses alone were responsible for up to seven percent of deaths in the U.S. per week in recent winter seasons. When it comes to diagnosing respiratory illnesses, there can be real challenges due to overlapping symptoms and distribution of diseases, which is why having a multi-target diagnostic can be particularly useful.  

For more about the importance of “Point of Need” diagnostics like those for SARS-CoV-2, influenza, and RSV, listen to this episode of “Science Diction” from MRIGlobal. 

Identifying the etiologic agent is critical in helping direct treatment of each of these diseases and preventing their spread. There are several platforms and targets that may be suitable for respiratory disease diagnostics. For COVID-19, influenza, and RSV, some of the most common in vitro diagnostics (IVD) typically fall into one of these three categories: 

  1. Immunoassays such as lateral flow assays for viral antigen detection.  
  2. Mass spectrometry such as breath tests to detect volatile organic compounds associated with viral infection. 
  3. Molecular tests may use PCR or LAMP-based assays to amplify viral genetic material. 

These platforms can use a variety of sample types, such nasal swabs and nasopharyngeal swabs, or even saliva, and they can require instrumentation like readers, or thermal cyclers for the molecular assays.  

Design and Development Process for IVD
The IVD design and development process typically takes place in several stages, with different activities designated during each stage. The gates indicate a stopping point at which your team should assess your readiness for the next stage.  

Stage 1: Initiation and Feasibility
The very first stage of development shown here is the initiation and feasibility stage. This is intended to assess the feasibility of completing the project. Activities may include preparation of an initial project proposal, establishing a business case or development of initial proof-of-concept assays and device prototypes. 

Stage 2: Concept and Definition
At this stage, it is important to consider details like the intended use of the assays being developed, the type of assay employed, the type of specimen testing, and the full end-to-end workflow approach for everything from sample prep to data analysis and reporting. Further market evaluation, customer and regulatory requirements, and prototype test development should be explored in this phase in addition to development a preliminary manufacturing plan and performing an intellectual property review. 

At this point in the process, there are also several questions that are important to answer. They are easily summarized as why, who, where, and what.  

Why Why is the first question to ask. What is the intended use of the final product you’re developing? Which diseases or infectious agents are being targeted?  

Who Who will be administering the assay? Will it be self-administered? Will it be administered by healthcare workers, or will it be administered by lab techs?  

Where Where will the assay be used? Will it be used at home and in a point-of-care setting or will it be used in a high-throughput hospital and testing lab?  

What? What type of assay is being developed? The specimen type and the end-to-end workflow components should also be considered.  

While these sound like simple questions, clarifying why, who, where, and what is critical in designing a promising product that fits the use case in mind and is imperative for the success of the final product. 

Assay Types and Workflow
The most common tests for respiratory tract infections are immunoassays and molecular tests. 

  • Immunoassays can be handheld lateral flow immunoassays. They can also be higher throughput enzyme-linked immunosorbent assays (ELISAs). 
  • For highly multiplexed tests, these are most often nucleic acid amplification tests like PCR and LAMP but can also include Sanger sequencing, fragment length analysis, and next-generation sequencing. 

In addition to thinking about the type of assay, it is also important to consider what the best specimen type is and what should be included on the label. If targeting lower respiratory infections, then those specimen types might include bronchoalveolar lavage, bronchial wash, tracheal aspirate, sputum, and lung tissue. If targeting upper respiratory infections, the specimen types are often nasal swabs, nasal aspirates, nasopharyngeal swabs, oropharyngeal swabs, and saliva. Many times, these swabs are also in a certain transport media, which must also be taken into consideration. 

Although developers like to focus on the type of assay, it is important to think about its development in the context of what the full workflow will look like. It is also important to know what sample collection is going to look like, including what the specimen type is going to be and what sample preparation process is optimal, the sample testing procedure, data analysis, and finally, the reporting process. All methods, reagents, consumables, equipment, and software that will be a part of that process should be considered before beginning the design and development phase. 

Stage 3: Design, Development, and Verification
During this stage, the design inputs and outputs may be further defined, and a final prototype device is developed and tested. The design verification process will confirm that the developed assay sufficiently meets product requirements and specifications. 

Principles of Molecular Assay Design
Target Selection and in silico Analysis
The first step of molecular assay design is selecting target genes – most importantly choosing sequences that are conserved in all strains of the pathogen you’re trying to detect – and are specific to that pathogen (i.e., not present in other microbes). In addition to selecting conserved genes, consider whether there are specific genes or markers of interest—for example, antimicrobial resistance markers, toxin genes, or other virulence factors that are going to be important to the clinician.  

Once target genes are selected, assay development can be moved to the primer and probe design stage. There are several considerations at this stage, including the sequence conservation and specificity, oligo interactions, the melting temperature of your oligos, oligo length, amplicon length, percent GC, fluorophore assignment, and much more. If a multiplex test is being developed, this becomes even more complex, as both intra- and inter-assay oligo interactions need to be considered, since these assays are all going to be together at the same reaction.  

During this process, more than one candidate assay per target gene should be designed. This de-risks the development process by allowing baseline feasibility tests of multiple assay options to be performed, then downselect only the candidate assays that perform best.  

An important part of the design process is in silico inclusivity and exclusivity testing—read more about this in “Bioinformatics in Assay Development.” This should be performed during the initial screening of designs and before the assay progresses to wet lab testing. If a multiplex test is being developed, then in silico testing needs to be performed with the understanding that all assays will be present in the same reaction. If you evaluate the assay designs independently rather than as a set, you may miss adverse oligo interactions or potential off-target detection involving oligos from different assay designs It is also important to note that in silico testing is only as good as the database being used, so having a well-curated database is essential.  

Initial Screening and Downselection
After designing candidate assays, an initial screening and downselection process should be performed to determine which of those candidate assays for each target are the most promising. This is done by performing baseline (pre-optimization) testing of the candidate design, including evaluating the approximate limit of detection (LoD) of the assay, linearity of the assay, inclusivity, and exclusivity. During this phase, the inclusivity and exclusivity testing is very small scale, so typically guided by the in silico results, and specifically investigating anything that was predicted to cause issues during in silico analysis.  

Assay performance metrics like amplification efficiency, relative fluorescence, and others should also be assessed. Initially this can be performed in singleplex, but if working with a multiplex assay, ultimately its performance in the final multiplex is what really matters.  

Five Keys to Remember in Assay Development

  1. Have a clear concept of the final product. That’s the why, who, where and what of your assay before initiating design and development.
  2. Leverage bioinformatics tools for assay design and in silico testing, making sure that you have a high-quality, well-curated database.
  3. Complete preliminary assay performance testing early – including feasibility testing of the full workflow with clinical specimens – before locking down the assay.
  4. Prepare a regulatory plan that is updated to track the evolution of your regulatory strategy and FDA feedback.
  5. Access the latest FDA guidance and publications to understand general requirements of your IVD and engage with the FDA early and often to get the specifics. 

Assay Optimization
After selecting the best performing candidates, assay development then proceeds to the optimization phase, where performance of those selected assays is improved. Assay optimization can encompass several variables, including the assay components themselves, the concentrations of those components, the cycling protocol, as well as other variables.  


Feasibility Testing
After optimization, selected assays progress to feasibility testing. This testing encompasses the full end-to-end workflow and should include clinical specimens, if possible, whether those are new, residual, or contrived specimens. The point of feasibility testing is to mimic the later analytical and clinical studies but on a smaller scale. The goal at this point is to screen the assay through small scale studies to confirm it is promising, and worth investing the time and money required for analytical and clinical studies. 

Stage 4: Validation and Transfer to Manufacturing
Assuming feasibility testing goes well, the developed assay(s) may then move into analytical and clinical validation studies, the specifics of which vary based on the type of assay and targets. Analytical validation typically includes a linearity test if the assay is quantitative or semi-quantitative, an LoD study that includes preliminary range-finding, a precision study, inclusivity, exclusivity, interfering substances testing, flex studies, and reagent and sample stability studies. Ultimately, with an established and validated manufacturing process, the assays will transition to GMP manufacturing.  

Stage 5: Regulatory

The importance of understanding and successfully navigating the regulatory process cannot be overstated. It is crucial to gaining approval for your IVD and eventual market access. Read more about this process at “Navigating Regulatory Challenges in Assay Development,” which provides insight into the necessary documentation, importance of early engagement with the FDA, and an understanding of regulations and standards.  

Stage 6: Product Launch
Congratulations! You’ve successfully reached your goal of being prepared to launch a novel disease assay onto the market!  


For additional expertise on this topic, MRIGlobal’s Yvette Girard, MPH, Ph.D., Joe Russell, Ph.D., and Jennifer Stone, MS, hosted a free webinar on the development of rapid, accessible, and portable infectious disease diagnostics for seasonal respiratory diseases like influenza, RSV, and SARS-CoV-2. Watch the webinar recording at “Infectious Disease Diagnostics for Influenza, RSV and SARS-CoV-2.”  

Person using a tissue


Contact MRIGlobal for further information about our work with infectious diseases. Through a multidisciplinary approach, we provide scientific and subject matter expertise for the development of medical countermeasures research against specific threats, while expanding and accelerating the delivery of high quality clinical diagnostic products.  

To discuss how we can help your project be successful, contact us today.  


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