Chorus SARS-CoV-2 test on saliva ("diagnostic chewing gum")

is aimed to the detection of total antibody (mainly IgG and IgA) in saliva. 

Under development by DIESSE Diagnostica Senese S.p.A., in collaboration with with the National Institute of Infectious Diseases (INMI) L. Spallanzani, Università degli Studi di Padova, and the Monoclonal Antibody Discovery (MAD) Lab of Toscana Life Sciences Foundation (TLS). 

Chorus SARS-CoV-2 test on saliva

DIESSE's commitment to the COVID-19 pandemic was born out of the scientific  collaboration with the National Institute of Infectious Diseases (INMI) L. Spallanzani  which had led to the development and subsequent experimentation and validation in Sierra Leone of 4 kits for the detection of IgG antibodies against the four Ebola virus serotypes (Zaire, Sudan, Bundibuyo and Marbourg). These donated kits were used by INMI researchers in a collaborative project with the Italian NGO EMERGENCY at the "Goderich Surgical Center" in Sierra Leone. The positive development of the EBOLA tests led to the development of diagnostic tests for the detection of IgG, IgA and IgM antibodies to SARS-CoV-2 with INMI.


This collaboration was based on the use of the SARS-CoV-2 Strain "2019-nCoV / Italy- INMI1" isolated in the INMI laboratories in February 2020 as native antigen for the development of the three ELISA kits for the detection of anti-SARS-CoV-2 IgA, IgG and IgM antibodies. The native antigen has been obtained from the virus cultivated, inactivated - through a proprietary and patented protocol - and purified in the BSL3 laboratories of DIESSE. These tests have been developed to provide results "aligned" with the IFA method developed in-house by INMI and used in the serological evaluation of COVID 19 patients. After a thorough evaluation of their performances, the newly developed ELISA kits have replaced the IFA method in the epidemiological surveillance and subsequent diagnosis phase at the INMI Spallanzani.


As the current SARS-CoV-2 pandemic continues, antibody assays are urgently needed for rapid diagnosis, contact tracing and for epidemiological studies. These tests should ideally have high level of sensitivity and specificity, to allow a reliable serological  diagnosis, be of simple and secure execution starting from the preanalytical phase to the final result recording and be widely available for mass screening of the population. Saliva has been increasingly used over the last few decades for evaluating human health  It is an integrated mixture of secretions of the different salivary glands, gingival crevicular fluid, and mucosal transudates.


The use of saliva has the following advantages:

 

  • It can be collected by the patient, even at home, minimizing the exposure of health care workers to nosocomial infections. This also reduces the need for health care personnel and waiting times for sample collection, resulting in easier crowd control regulations in clinical settings and thus avoiding further virus transmission.
  • It is easily accepted by the patients since it is non-painful and non-stressful. Therefore, it can be used for serial samplings and in large scale or epidemiological studies, being especially advantageous in certain populations, such as children.
  • It is easy, fast, and cheap to collect, allowing widespread testing.

 

Standardization of the collection of the saliva sample is of primary importance to obtain a suitable sample, rich in gingival crevicular fluid, whose composition resembles that of serum, and that is richer in antibody compared to the other oral fluids. For this reason, we propose the use of a device composed of a special collection tube and of an absorbing pad. No particular preparation of the patient is required: it is sufficient not to have assumed food or beverages thirty minutes before collection. The absorbing pad has to be kept in the mouth between the gingiva and the cheek for one minute and then it has to be put in the tube, that can be sent to the laboratory. Saliva is stable for five days at room temperature and therefore no particular shipping conditions have to be maintained.  Once in the laboratory, the tube is centrifuged for a few minutes and the obtained oral fluid is ready for testing.


The new “Chorus SARS-CoV-2 Screen Test” designed by DIESSE is aimed to the detection of total antibody (mainly IgG and IgA) in saliva. The test, based on the competition method, exploits a human recombinant monoclonal antibody raised against the S1 subunit of the Spike protein conjugated with horseradish peroxidase tracer that is mixed with the saliva sample in the reaction well.


The test's uniqueness is the native antigen. The native antigen, used in solid phase of the reagent, is obtained from VERO E6 cells infected with the isolated strain 2019-nCoV/Italy-INMI1 through a patented protocol of inactivation and purification in BSL 3 laboratory of DIESSE.


It is performed in complete automation in the Chorus instrument, by means of single test, ready-to-use dedicated devices based on the ELISA method, and the results are obtained in just about 30 minutes. Chorus represents a reliable laboratory solution able to provide quality results, offerin high sensitivity and specificity and efficiency with an easy to use automated method. Moreover, the new test can be easily converted to the common 96 microwells ELISA plate format, compatible to all the automated microplate processors available in the market, thus allowing the screening of a large number of samples.


The test is developed in cooperation with Professor Mario Plebani’s group at the University of Padova, where the validation of the kit using samples from patients atvarious stages of the disease is ongoing.

diagnostic chewing gum - making testing more accessible, and results more accurate

our insight of the week -  September 20, 2020
photo courtesy company
Back in February, when large parts of the world still felt like the novel coronavirus was not their concern, Massimiliano Boggetti was already working on a test kit. Boggetti is the CEO of the Italian diagnostics company DIESSE based in Tuscany. They had previously collaborated with the National Institute for Infectious Disease Lazzaro Spallanzani in Rome to produce a test kit for Ebola. Now, the same institute collected Covid-19 samples isolated from the Chinese patients who were the first to arrive in Italy with the virus. “When Covid-19 appeared, it was very clear to us that we wanted to contribute to the fight against it”, says Boggetti. 

In general, there are molecular tests to detect genetic material from the virus and serological tests to detect antibodies which indicate the body’s immune response. The molecular tests show an early stage of the virus entering the body – a presence of the virus and an active infection – while the serological tests work post-infection and show a current or past exposure to the virus. 
photo courtesy company 
Boggetti and his team at DIESSE deactivated the virus, which means it can no longer cause an infection, and then used it to detect the possible antibodies produced against the virus. With this method, they were able to launch an ELISA serological test which is a commonly used analytical biochemistry assay based on a native antigen in April. “It resulted in a very specific and accurate test”, says Boggetti. It is able to show whether or not someone has been exposed to the virus and has created an immune response.

Furthermore, this was one of the first test kits to detect the various serum antibody isotypes: IgG, IgA and IgM. This means that the kits can test for infection over a wide time span. IgM-antibodies are produced at the early stage post-infection and therefore are a marker for the acute phase. For respiratory viruses like the SARS-COV-2, IgA-antibodies are also a marker for the early acute phase of infection, since they are produced in the respiratory mucus. IgG-antibodies are produced at a later stage. With its wide range, the test kit could be used by clinicians to follow the evolution of a patient’s infection.
photo courtesy company 
Now, DIESSE started to work on the next generation of tests. “The idea was to produce a test that can be used by anybody, with very basic instruments”, says Boggetti. The tests will be based on saliva, as opposed to DIESSE’s previous tests that needed blood and most of the other tests currently on the market that require a more invasive nasal and throat swab. 

Boggetti hopes they will be able to launch this test in September. “The idea is that when you come back from holidays, you can find out if you have been exposed to the virus through the saliva test”, says Boggetti. All it requires is for the person to chew on a small sponge and spit it into a test-tube. This is then sent to a laboratory and there, after centrifugation of the samples to collect the saliva, scientists simply need to put the sample into DIESSE’s automated system. “The machine will do the rest and provide the results”, says Boggetti.

Through this simple method, Boggetti hopes to rule out a lot of potential sources of error. “The majority of molecular testing arrives at 70 percent specificity, also because the collection method is so complicated that it can create a lot of variables in the results”, says Boggetti. “Our goal is to come up with something even more convenient than molecular testing without giving up accuracy.”

DIESSE also started a collaboration with Toscana Life Sciences and Rino Rappuoli to include a dedicated test for the therapeutic they are currently developing.

Until a vaccine or therapeutic will be available, the best approach to fight the pandemic is to intensify testing, says Boggetti. “We need to trace the virus to stop it from spreading”, he says. 

September 20, 2020 by kENUP