Point-of-care isothermal molecular test system detecting SARS-CoV-2
Testing for the SARS-CoV-2 infection currently has a significant impact on clinical laboratories globally concerning capacity building; scale-up of testing, stock-out of laboratory supplies, and high cost of reagents. The Nucleic Acid Amplification Test (NAAT) recommended by the World Health Organization (WHO) for the routine confirmation of COVID-19 requires expertise in molecular biology techniques and is expensive to implement. Presently, there is a global shortage of RNA extraction kits and enzymes used in nucleic acid testing. This global shortage further increased the cost of these materials, thereby exerting a severe economic toll on developing countries, especially in Africa. There is a need to create an alternate NAAT that will be affordable, accessible, medium-throughput, and provide fast results without compromising test sensitivity, specificity, and accuracy.
Our solution involves the use of diverse nucleic acid preparatory systems, especially those accessible and not in global shortage, to get the sample ready for testing. This involves the use of routine RNA extraction methods were available, a highly affordable automated sample extraction system, and ultimately a sample lysis and release (SLR) buffer. The nucleic acid SLR buffer in development will lyse and release the RNA. The buffer offers a fast and affordable way for RNA preparation compared to the conventional RNA extraction method and will not require specialised expertise nor equipment. The SLR further contains enhancers improve PCR amplification and can be utilised for point-of-care (POC) or laboratory testing. The target is sample preparation within 5 - 30 minutes.
The RNA sample will be tested using our isothermal molecular test for SARS-CoV-2. Based on accessible isothermal technology, reagent, and equipment, our assay for SARS-CoV-2 is being validated in-house. The assay’s limit of detection (LOD) is currently 100 copies of the plasmid template and 10,000 copies/µL of SAR-CoV-2 RNA from the clinical sample. We are optimizing the enzymes for reverse transcribing the RNA to further reduce the LOD for clinical samples and incorporate the simultaneous detection of a human housekeeping gene as an internal assay control. Ultimately, the assay will be a multiplex isothermal test concurrently detecting SARS-CoV-2 and Influenza A, as the first in our intended syndromic diagnostic assays for respiratory diseases. Initial assay development was supported by a grant from FATE Philanthropy Coalition for COVID-19 (FPCC) worth $45,000.
The isothermal assay is based on the Recombinase isothermal technique, coupled with fluorescent detection to achieve medium throughput. We earlier exploited its potential for POC use to develop a multiplex molecular diagnostic for febrile illnesses. Within 30 minutes, six multiplex tests differentially detect the top 10 causes of febrile illness in children, including malaria (Plasmodium falciparum), typhoid Fever (Salmonella enterica serovar typhi and paratyphi), Streptococcus pneumoniae, Brucella abortus, Leptospira interrogans, Dengue 1-3 viruses, Yellow fever virus, Rotavirus A, Chikungunya virus and Lassa fever virus. These Febrile illness syndromic assays are undergoing field testing. Thus, we intend to utilize the same isothermal platform to develop the respiratory disease syndromic assays, starting with one multiplex test for SARS-CoV-2 and Influenza A viruses.
- Develop a multiplex isothermal assay for SARS-CoV-2 and Influenza viruses. We have designed and are evaluating primers and probes for the multiplex recombinase molecular assay. Assay performance will be evaluated according to the World Health Organization (WHO) prequalification standards, being a WHO Prequalification Testing Laboratory. It will also be evaluated by stakeholder agencies in the Nigerian health sector.
- Identify and implement sample preparatory systems suitable for diverse settings such as POC and field testing. We will identify and implement sample preparatory methods, for different cost categories, to accommodate use with existing facilities and facilitate uptake even with minimal funds.
- Implement and evaluate the utility of non-fluorescent detection systems, such as disposable Biotin-dual-labelled lateral flow strips and/or cartridges, to match lower throughput settings.
- Explore public-private partnerships to develop the entire respiratory disease syndromic assays, other assay configurations, and set-up local manufacturing facility with support from reputable institutions such as Merck OEM. Currently, we are utilizing commercially recombinase reagents. A manufacturing facility will truly customize these assays and reduce hands-on steps and technical expertise required.
- A molecular test system amenable for point-of-care detection of SARS-CoV-2 virus and deployable to remote areas of resource-constrained countries
- Multiplex assays optimizable for local health indices, accounting for the co-infections and multi-morbidities that frequently coexist. Consequently, these solutions have utility for routine testing post-COVID 19, e.g. for syndromic febrile illness assays
- Eliminate presumptive therapy and preserve antimicrobials
- Ensure the correct treatment first time, truly personalize therapy, and improve health indices.
- Comparatively more affordable than regular NAAT assay. Currently, our molecular assay costs $10 while the regular NAAT test can cost $25 – 125. Ultimately our solution of SLR buffer and isothermal assay should cost $10 per test when locally manufactured.
- Comparatively more affordable and portable equipment than regular real-time PCR systems. Isothermal equipment with field casing is cheaper ($11,050) than conventional real-time PCR systems ($20,000-50,000).
- Our molecular test system can be optimized to field test 32 samples within 40-60 minutes, providing medium throughput. With RNA extraction, conventional NAAT can take 2 – 5 hours for 1-96 samples.
- Currently utilizing commercial reagents and equipment. Thus, the sourcing of materials and isothermal equipment may be impacted by global travel restrictions.
- Lack of oligosynthesizer to make our primers and probes in-country
- Lack of an accredited diagnostics manufacturing facility in-country
- Biosafety and biosecurity precautions for field testing pathogens with aerosol transmission
Stage of development
With support from FATE Philanthropy Coalition for COVID-19 (FPCC), the SARS-CoV-2 assay has been developed and demonstrated, while we are tweaking challenges with the Influenza A assay. Different formulations and versions of our nucleic acids SLR buffer are being evaluated with progressively better results.
In addition, MTN Nigeria Foundation had partnered with NIMR to procure and install an oligosynthesizer capable of meeting our needs for primers and probes in Nigeria and potentially the West African sub-region. Hopefully, this will be finalised in the next 3-6 months.
Next steps will involve:
- Pilot field testing of the SARS-CoV-2 isothermal test system in Lagos Nigeria. We await feedback on a proposal to African Development Bank to finance the field testing.
- Bulk procurement of isothermal equipment, enzymes, and reagents
- Support to develop the entire Respiratory Diseases Syndromic Assays and implement other assay configurations
- Support to set-up a diagnostics manufacturing facility. Discussing technical support with Merck OEM Unit