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viernes, 3 de julio 2020
03/07/2020
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UdeA Researchers Designed a Molecular Diagnostic Protocol for SARS-COV-2

By: Jennifer Restrepo de la Pava — Journalist

Researchers at Universidad de Antioquia designed and developed Colombia Protocol, which proposes a new organized workflow to support the country in the detection of the virus SARS-COV-2, which causes COVID-19.

"The novelty about Colombia Protocol is that it is not a simple diagnostic methodology. It includes a series of molecular, experimental, and complementary approaches, used either combined or independently, according to the local conditions, allowing the detection of SARS-COV-2 at levels of specificity and sensitivity that are equal to or higher than those reported using the Berlin Protocol. This results in a more accurate diagnosis,” said the head of the research Gustavo Adolfo Gámez de Armas, Professor of the School of Microbiology.

But, what is the main difference between the Colombia Protocol and the existing protocols? To diagnose the virus in the country, the real-time PCR molecular test (qPCR) is currently performed. This test usually follows three protocols: Institute Pasteur Paris, China CDC, and Berlin Protocols. The latter is recommended by the World Health Organization (WHO).  

According to researchers, these protocols emerged early within the pandemic when little was known about the virus, and therefore there was not enough information about its genome and characteristics.
“The available protocols are essentially aimed at detecting E, N and RDRP genes, related to this and other existing coronaviruses. However, Colombia Protocol is aimed at genomic sequences of the S gene, encoding the Spike protein, which according to bioinformatics analyses developed by Professor Gámez, are exclusive sequences of the SARS-COV-2 virus,” as explained by Professor Andrés Felipe Zuluaga Salazar, a project co-investigator and coordinator of the Integrated Laboratory of Specialized Medicine (LIME by its Spanish acronym) of the School of Medicine.  

The reduction in testing time is key to this new proposal “the time required using the Berlin Protocol is longer as compared to the model we propose. Berlin protocol requires three consecutive qPCR tests to provide a diagnosis; on the contrary, Colombia Protocol shortens this procedure by performing only one test promoting the use of fewer resources,” said Professor Carlos Muskus López, co-investigator and member of the Programs for Study and Control of Tropical Diseases Group (PECET by its Spanish acronym), from the School of Medicine.

Colombia Protocol includes biologists, microbiologists, doctors, bacteriologists, engineers, students and non-teaching administrative staff of UdeA. Reference image.

Advantages of the Colombia Protocol

Identifying a unique genetic sequence of the virus opened the path for faster, more economical, specific and sensitive detection of the virus, expanding testing coverage. According to Professor Gámez, sensitivity and specificity are the two major differences in Colombia Protocol. Besides, they contribute to fight against cross-contamination problems in the laboratory due to the coverage expansion.

Sensitivity accurately detects low numbers of copies of the virus in the sample “in the standardization of the test, there is a parameter called analytical sensitivity, that is, the minimum amount of viruses detected using the diagnostic protocol. The higher the sensitivity, the higher the probability of finding the virus in a sample of a person with a low viral load,” added co-researcher Carlos Muskus.

Meanwhile, specificity allows us to obtain unambiguous positive results for each sample analyzed. “As it is based on a molecular design of double-mismatch allele-specific qPCR, Colombia Protocol allows more precisely to discriminate the SARS-COV-2 virus from other human and animal coronaviruses, which could be generating false positives in the tests. In addition, an earlier detection of the same, even before the onset of the first symptoms, also makes it possible to detect asymptomatic cases,” added Gustavo Gámez, who is also a member of the Genetics, Regeneration and Cancer Group of the School of Exact and Natural Sciences.

For the design Colombia Protocol, information taken from the bioinformatic analysis of 3000 genomic sequences reported in the GISAID (Global Initiative on Sharing All Influenza Data) was used as input for the manual and detailed design of qPCR Primers. This allowed comparing the method with existing approaches within the development stage, based on the Berlin Protocol: commercial kits and home tests being used in Colombia.

Expanding molecular testing will facilitate more in-depth population and epidemiological studies for decision-making. Reference image.

Being a versatile model, Colombia Protocol will not depend much on the import of supplies needed to carry out the tests. These include primers, probes and enzymes which, in addition to being expensive, take 30 to 45 days to arrive in the country.

This proposal emphasizes the use of human talent and installed capacity: laboratories, equipment, networks and available inputs. The tests suggested in Colombia Protocol could be performed in a larger number of molecular biology laboratories in Colombia, Latin America, and the world.

“There are not many machines here for real-time qPCR testing used in the country, but there are many conventional qPCR thermal cyclers in different molecular biology laboratories. That is why the response to the tests in Colombia is so limited, unlike countries with much more capacity,” explained Gustavo Gámez.

For implementing the Colombia Protocol, the isolation of the SARS-COV-2 virus developed by the Immunovirology Group of the School of Medicine last April was very significant. This allowed Professor Gámez to have the positive control necessary to develop the Protocol.

The researcher Wbeimar Aguilar Jiménez, collaborator of the Protocol and member of the Immunovirology Group, pointed out that the next stage is to extrapolate the tests carried out in the laboratory and obtain institutional guarantees “in addition to the  test accreditation, there are other variables that do not depend on the University, such as permits and the authorization by INVIMA (Colombia’s regulatory agency), the Ministry of Health and Social Protection and the National Institute of Health,” Aguilar added.

Researchers from Universidad de Antioquia call on the National Government to analyze this new protocol that would eventually support the expansion of COVID-19 testing in the country, bearing in mind that its implementation is based on the use of the installed capacity of molecular biology laboratories (fixed or mobile) in the country and that do not require a lot of economic resources. It is expected that Colombia Protocol will contribute to solutions to some problems caused by the pandemic and will have a national, regional, and international impact.

Figure 1. Results laboratory tests: detection of the virus by qPCR, Colombia Protocol matching the Berlin Protocol. (Courtesy image- project)

Figure 2. Laboratory test results: detection of the virus by conventional PCR, Colombia Protocol surpassing the Berlin Protocol. (Courtesy image- project)

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