WO2022101466A1 - Detection of viral nucleic acids from soiled disposable tissues - Google Patents

Abstract

The invention relates to a method for self-collection of biological samples by nose-blowing and/or expectoration, in order to detect viral nucleic acids and diagnose viral respiratory infections. The invention also relates to a method for detecting nucleic acids of a respiratory virus from a soiled tissue and a method for diagnosing a viral respiratory infection in an individual who has provided the biological sample by nose-blowing and/or expectoration. The invention also relates to a kit for collecting a biological sample.

Description

Detection of Viral Nucleic Acids from Soiled Disposable Tissues

Parent Patent Application

This patent application claims priority from French patent application number FR 20 11647 filed November 13, 2020. The contents of the French patent application are incorporated by reference in their entirety.

Technical area

The present invention relates to the field of the detection of respiratory viruses. In particular, it provides a method for collecting biological samples for the diagnosis of viral respiratory infections.

Background of the invention

Respiratory viruses are the most common pathogens in humans. Viral respiratory tract infections affect millions of people annually and are a major cause of morbidity and mortality, particularly among those at risk - young children, the elderly, immunocompromised patients and those with lung disease. or underlying cardiac. For example, each year, influenza causes respiratory tract infections in 5 to 15% of the world's population with serious illness in approximately 5 million people and 290,000 to 650,000 deaths (who.int/fr/news-room/ fact-sheets/detail/influenza-(seasonal)). Similarly, respiratory syncytial virus (RSV), which is the most common cause of respiratory infections in young children worldwide, causes nearly 34 million episodes per year (Ducharme, BMJ, 2011, 342: dl658). The common characteristic of viral respiratory infections is their high contagiousness, which explains the risk of occurrence of severe epidemics. The fight against respiratory viruses has therefore become a major issue in terms of public health, all the more so since, worryingly, new respiratory pathogens are emerging. Indeed, the epidemics of Severe Acute Respiratory Syndrome in 2002-2003 (due to a coronavirus, the SARS-CoV virus), avian influenza A (H5N1) in 2004, influenza A (H1N1) in 2009-2010, Syndrome Middle East respiratory disease (due to coronavirus MERS-CoV) in 2012, and avian influenza A (H7N9) in 2013-2014, as well as the pandemic that started in China in 2019 and

CORRECTED SHEET (RULE 91) ISA/EP which is caused by a new coronavirus, the Severe Acute Respiratory Syndrome coronavirus 2 or SARS-CoV-2, have reminded and continue to remind of the frequency and severity of acute respiratory infections around the world.

Clinicians and virologists are now convinced of the importance of making an accurate diagnosis of viral respiratory infections. However, these infections are currently misdiagnosed because they are community infections for which a wait-and-see attitude is generally the most widespread diagnostic and therapeutic attitude. The major problem facing the practitioner lies in the multiplicity of respiratory pathogens likely to be responsible for the same symptomatology (Brunstein et al., Diagn. Mol. Pathol., 2006, 15: 169-173). The availability of a test that can very quickly identify the virus in question is essential for several reasons. Curative treatments, when available, are only effective at an early stage of viral respiratory infection. Properly detecting viral infections is also essential in the context of the fight against antibiotic resistance, because being able to confirm that the infection is not of bacterial origin makes it possible to limit the unnecessary prescription of antibiotics (Byington et al., Arch Pcdiatr.Adolesc.Med., 2002, 156: 1230-1234; Henrickson, Pediatr., 2005, 34: 24-31; Gonzales et al., Clin. Infect. Dis., 2001, 33: 757-762; Keske et al., Eur. J. Clin. Microbiol. Infect. Dis., 2018, 37: 779-783; Mahony et al., J. Clin. Microbiol., 2009, 47: 2812-2817; Rogan et al., I. Mol. Diagn., 2017, 19: 460-467). The accurate and timely identification of respiratory viruses is also essential for the adoption of public health measures and for the control of possible outbreaks.

The diagnosis of viral respiratory infection is usually based on a nasopharyngeal sample obtained by swab and transported in a viral transport medium to a biological analysis laboratory. Upon receipt, the laboratory may choose to apply different technologies, such as sensitive detection of viral antigens (by immunofluorescence or immunochromatography) and detection of viral nucleic acids (based on real-time PCR with or without preceded reverse transcription). The quality of the sample is the essential condition for any reliable virological diagnosis. However, a nasopharyngeal swab requires the intervention of qualified medical personnel because the swab must be inserted deep into the nostrils up to the nasopharynx. Inadequate swabbing can lead to false negative results.

CORRECTED SHEET (RULE 91) ISA/EP There is therefore a need for new simplified but reliable strategies for collecting biological samples for the diagnosis of viral respiratory infections.

Summary of the invention

The present invention provides an alternative method comprising self-collection of a biological sample for the detection of respiratory viruses. More specifically, in the proposed method, the individual to be tested collects the sample by blowing his nose and/or spitting into a disposable handkerchief. The soiled tissue is inserted into a container, which is then transported to an analysis laboratory, where it is used to detect viral nucleic acids using a sensitive molecular method. The experimental results presented below and obtained, both in a crèche over the course of a year and in two classes of small and large sections of a nursery school, show that the method described here allows the effective detection of seasonal respiratory viruses. and respiratory viruses. In addition, experimental results obtained using soiled tissues by adults presenting for a diagnosis of COVID-19 by a standard nasopharyngeal swab have highlighted the promising performance of the approach of the present invention in the detection of the SARS-CoV-2. The collection of the biological sample proposed here is a simple, rapid, and non-invasive act. It does not require any medical skills and makes the patient autonomous. It reduces the possibilities of external contamination and eliminates any risk of contamination from a third party (by eliminating any movement of the patient and/or any contact with a healthcare professional at the time of sample collection). In addition, compared to other methods for collecting biological samples, the method proposed here is inexpensive.

Accordingly, the present invention relates to a method for detecting nucleic acids of a respiratory virus from a handkerchief containing a biological sample of an individual to be tested, the method comprising the following steps: obtaining a handkerchief containing a sample biological, where the biological sample has been collected by a method comprising the following steps in which: the individual to be tested blows his nose in a single-use handkerchief;

CORRECTED SHEET (RULE 91) ISA/EP the individual introduces the soiled handkerchief alone into a container and closes the container; and the container containing the soiled handkerchief is sent to a biological analysis laboratory; recovering the biological sample contained in the soiled handkerchief using a physiological solution, in particular a saline phosphate buffer; detecting the presence of viral nucleic acids in the recovered biological sample; and if viral nucleic acids have been detected, concluding that the respiratory virus is present in the biological sample and possibly diagnosing a viral respiratory infection in the individual having collected the biological sample.

In some embodiments, the biological sample collection method further includes a step in which the individual to be tested spits into the single-use handkerchief.

In some embodiments, the container has a volume of 50 ml.

In some embodiments, the container is a plastic centrifuge tube or a plastic needleless syringe.

In some embodiments, the soiled handkerchief is transported to a biological analysis laboratory at room temperature.

In some embodiments, the container containing the soiled tissue is inserted into an envelope and transported to a biological analysis laboratory by mail or by specialized medical transport.

In some embodiments, the respiratory virus is selected from the group consisting of influenza viruses, parainfluenza viruses, respiratory syncytial virus (hRSV), metapneumovirus (hPMV), adenovirus, rhinovirus, and coronavirus.

In some embodiments, the respiratory virus is selected from the group consisting of influenza A viruses, including HINT, H3N2, A(H5N1), A(H7N7), A(H9N2), and A(H7N9) subtypes. ), influenza B viruses, parainfluenza viruses type 1, type 2, type 3 and type 4, respiratory syncytial virus (hRSV) subgroup A, respiratory syncytial virus (hRSV) subgroup -group B, metapneumoviruses subtype A, metapneumoviruses subtype B, adenoviruses

CORRECTED SHEET (RULE 91) ISA/EP responsible for respiratory infections, and the coronaviruses 229E, NL63, OC43, HKU1, B814, SARS-CoV, MERS-CoV, and SARS-CoV-2.

In some embodiments, the individual to be tested is suspected of being infected with a respiratory virus or is at risk of being infected with a respiratory virus.

In some embodiments, the individual to be tested is not a pediatric patient.

In certain embodiments, detecting the presence of viral nucleic acids in the recovered biological sample is carried out by a nucleic acid amplification technique, in particular a technique chosen from the techniques of PCR, RT-PCR, NASBA, TMA , LAMP, SDA, LCR and MLPA.

In another aspect, the present invention relates to a method of collecting a biological sample for the detection of a human respiratory virus in an individual to be tested, the method comprising the following steps in which: the individual to be tested blows his/her nose and /or spits into a disposable handkerchief; the individual introduces the soiled handkerchief into a container and closes the container; and the container containing the soiled handkerchief is taken to a biological analysis laboratory.

In embodiments where the biological sample is collected by spitting, the individual preferably has a productive cough.

In some embodiments, the respiratory virus to be detected is a respiratory virus selected from the group consisting of influenza viruses, parainfluenza viruses, respiratory syncytial virus (hRSV), metapneumovirus (hMPV), adenovirus, rhinovirus , and coronaviruses. For example, the respiratory virus can be chosen from the group consisting of influenza A viruses, in particular the H1N1, H3N2, A(H5N1), A(H7N7), A(H9N2), and A(H7N9) subtypes, influenza B viruses, parainfluenza viruses type 1, type 2, type 3 and type 4, respiratory syncytial virus (hRSV) subgroup A, respiratory syncytial virus (hRSV) subgroup B, metapneumoviras subtype A, metapneumoviras subtype B, adenoviruses responsible for respiratory infections, and coronaviruses 229E, NL63, OC43, HKU1, B814, SARS-CoV, MERS-CoV, and SARS -CoV-2.

CORRECTED SHEET (RULE 91) ISA/EP In some embodiments, the individual to be tested is suspected of being infected with a respiratory virus or is at risk of being infected with a respiratory virus.

In some embodiments, the individual to be tested is not a pediatric patient.

In certain embodiments, the soiled tissue is introduced alone into the container.

In some embodiments, the container has a volume of 50 mL.

In some embodiments, the container is a plastic centrifuge tube or a plastic needleless syringe.

In some embodiments, the container containing the soiled handkerchief is transported to a biological analysis laboratory at room temperature.

In some embodiments, the container containing the soiled tissue is inserted into an envelope and transported to a biological analysis laboratory by mail or by specialized medical transport.

The present invention also relates to the use of a biological sample obtained by a collection method described above, for the detection of nucleic acids of a respiratory virus and/or for the diagnosis of a viral respiratory infection in individual who provided the biological sample.

The present invention also relates to a method for detecting nucleic acids of a respiratory virus from a soiled handkerchief, the method comprising the following steps: recovering the biological sample contained in a soiled handkerchief, the soiled handkerchief having been obtained by a method for collecting a biological sample as defined in any one of claims 1 to 10; detect the presence of viral nucleic acids in the recovered biological sample; and if viral nucleic acids have been detected, conclude that the respiratory virus is present in the biological sample and possibly diagnose a viral respiratory infection in the individual who collected the biological sample.

CORRECTED SHEET (RULE 91) ISA/EP The terms “respiratory virus” and “human respiratory virus” are therefore used interchangeably herein. In humans, these viruses are transmitted by aerosols or droplets emitted either directly by coughing or sneezing or indirectly by contact via contaminated hands in contact with secretions.

Respiratory viruses to which the present invention may be applied include, without limitation, influenza viruses, parainfluenza viruses, respiratory syncytial virus, metapneumoviruses, adenoviruses, rhinoviruses, and coronaviruses.

In certain embodiments, the present invention is applied to the detection of an influenza virus. Influenza (influenza) viruses are enveloped RNA viruses belonging to the Orthomyxoviridae family. There are three types of influenza virus that infect humans: influenza A viruses, influenza B viruses, and influenza C virus. Type A viruses infect humans and many animal species: the avian species that make up the reservoir as well as different species of mammals, in particular the pig. Type A viruses (the most frequent and the most virulent) are even more finely characterized and differentiated into subtypes on the basis of their surface antigens: hemagglutinin (H1 to H18) and neuraminidase (NI to Ni l ). In humans, there are Hl, H2, H3 and NI or N2 viruses responsible for the annual flu. The H1N1 and H3N2 subtypes have been circulating most frequently in recent years. The pathogenicity of these viruses varies from year to year and depends on many factors including the age of the infected person, their vaccination status and the capacity of their immune system. In recent decades, cases of infection with avian and swine influenza viruses, including subtypes A(H5N1), A(H7N7), A(H9N2), and A(H7N9), have been reported in 'male. Human cases of infection with these subtypes are mostly associated with direct contact with animals or contaminated environments, but they do not result in effective human-to-human transmission. Influenza B viruses infect almost exclusively humans. There are two lineages of type B viruses: the B/Yamagata lineage and the B/Victoria lineage. Cases of influenza B virus infection are generally less frequent than those associated with influenza A virus. Types A and B viruses are responsible for seasonal epidemics, but only type A influenza viruses have been identified. origin of pandemics. The influenza C virus seems to be linked to sporadic cases and most often gives moderate flu expression.

CORRECTED SHEET (RULE 91) ISA/EP - 10 -

In certain embodiments, the present invention is applied to the detection of a parainfluenza virus. Parainfluenza viruses are enveloped RNA viruses that belong to the Paramyxoviridae family. In humans, four representatives of these viruses are found in the context of community viral infections: parainfluenza viruses type 1 and type 3 (which are classified in the genus Respirovirus) and type 2 and type 4 (which classify in the genus Rubulavirus). Parainfluenza viruses are transmitted through the air and cause a febrile reaction after 3 to 5 days of incubation. Type 1 and 2 parainfluenza viruses are limited to laryngeal involvement and infection occurs in children under 6 years of age. Type 3 parainfluenza virus spreads through the bronchi and bronchioles. The infection is early and appears before the age of two years. Type 4 parainfluenza is not very virulent. Since infection with parainfluenza viruses is not immunizing, reinfections are frequent throughout life. They can cause pneumonia in adults, especially in immunocompromised subjects.

In certain embodiments, the present invention is applied to the detection of a respiratory syncytial virus (VRS or hRSV for human Respiratory Syncytial Virus). hRSV is an RNA virus classified as a pneumovirus. Two subgroups A and B have been identified. hRSV is the most common cause of respiratory infections in young children worldwide. Highly contagious, this virus mainly infects infants under the age of two. An hRSV infection in young children can become complicated and become bronchiolitis, which can lead to hospitalization in exceptional cases. Reinfections are common. In adults, hRSV infection is rare, benign (except in the elderly), and is responsible for rhinitis or a flu-like syndrome.

In certain embodiments, the present invention is applied to the detection of human metapneumovirus (hMPV). This virus is a Pneumovirinoe identified in 2001, and whose genome is similar to that of the human respiratory syncytial virus. Phylogenetic analysis of hMPV demonstrated the existence of two major genetic lineages termed subtype A and B, which contain subgroups Al, A2 and B l, B2 respectively. Human metapneumovirus is responsible for upper and lower respiratory infections in young children, but also in healthy adults and immunocompromised subjects. This virus mainly causes infantile bronchiolitis, pneumonia and otitis and is thought to be responsible for 5% to 10%

CORRECTED SHEET (RULE 91) ISA/EP - 11 - pediatric hospitalizations for respiratory infections. Primary hMPV infection occurs in early childhood and is followed by multiple reinfections in adulthood.

In certain embodiments, the present invention is applied to the detection of an adenovirus, in particular a human adenovirus causing a respiratory infection. Adenoviruses are DNA viruses. There are 7 species of human adenoviruses (A to G) and 57 serotypes. Different serotypes are associated with different pathologies. Respiratory diseases are mainly associated with species B and C, and in particular with serotypes Cl, C2, B3, C5, B7, B 14, and B55 (pneumonia and other acute respiratory diseases), with serotypes Cl, B3, C5 and B7 (upper respiratory tract infections), and serotypes B3, E4, B7, and B21 (lower respiratory tract infection). Symptomatic adenovirus infections occur mainly in children. In immunocompetent patients, most infections are asymptomatic. However, adenoviral infections are increasingly recognized as causes of serious respiratory disease in immunocompromised adults.

In certain embodiments, the present invention is applied to the detection of a rhinovirus. Rhinoviruses are a species of virus belonging to the Picomavirus family. They are the main causative agent of colds and rhinitis in humans.

In certain embodiments, the present invention is applied to the detection of a coronavirus. Coronaviruses (CoV) are enveloped RNA viruses that make up the Orthocoronavirinae subfamily of the Coronaviridae family. Bats and birds, as warm-blooded flying vertebrates, are the ideal hosts for coronaviruses ensuring the evolution and dissemination of the coronavirus. Coronaviruses are normally specific to an animal taxon as a host, but sometimes these viruses can change hosts as a result of mutation. Their human-to-human transmission occurs mainly by close contact via respiratory droplets generated by sneezing and coughing or more rarely by indirect contact (hand-carrying). Seven main coronaviruses are generally cited as being able to infect humans (CDC, “Coronavirus - Human Coronavirus Types”, February 27, 2020). An eighth has been identified: B814 (Kapikian, Developments in Biological Standardization, 1975, 28: 42-64) - the first human coronavirus identified - but this strain seems to no longer be circulating. Four circulating coronaviruses are

CORRECTED SHEET (RULE 91) ISA/EP - 12 - considered not serious: 229E, NL63, OC43 and HKU1, which are thought to be the cause of 15 to 30 of common colds. More recently, three types of coronavirus responsible for severe pneumonia have been identified: SARS-CoV, the pathogen of severe acute respiratory syndrome (SARS) which appeared in 2002, MERS-CoV, the pathogen of the Middle East respiratory syndrome in from 2012, and SARS-CoV-2, the pathogen of coronavirus disease 2019 (Covid-19) which appeared in China in 2019 and responsible for a severe pandemic in 2020.

As will be recognized by those skilled in the art, the present invention can be applied to any other human respiratory virus which is not specifically listed here, in particular viruses transmissible by the respiratory route such as cytomegalovirus (CMV), varicella virus -shingles (VZV), Epstein-BaiT virus (EBV), parvovirus B 19, measles, mumps or rubella virus.

B. The Patients Concerned

In a method according to the invention, the patient collects the biological sample. The terms “person”, “individual”, and “patient” are used interchangeably herein and refer to a human being who may be infected with a respiratory virus, but who is not necessarily infected with that virus. These terms do not designate a particular age and therefore include infants, children, adolescents, and adults, including the elderly. In certain embodiments, the present invention applies to an individual who is not a pediatric patient (i.e. who is not a child, the child being defined in law as any elderly subject under 18 according to article ¹ of the Convention on the Rights of the Child).

In some embodiments, the individual is suspected of being infected with a respiratory virus or is at risk of being infected with a respiratory virus. A person can be "suspected of having a respiratory virus infection" if the person has clinical symptoms a priori attributable to a viral respiratory infection. Symptoms of a respiratory tract infection include, but are not limited to: fever, inflammation of the respiratory lining, coughing, sneezing, runny nose, stuffy nose, scratchy or sore throat, headache, muscle pain, chest pain, wheezing, fatigue, etc. A person can be considered “at risk of being infected with a respiratory virus” if there are cases of infection (or even an epidemic) in the area, the

CORRECTED SHEET (RULE 91) ISA/EP city, or the place where she lives, or because she has been in contact with an individual who has been diagnosed with the respiratory virus, or because in the case of this person the exposure to Respiratory virus has a higher likelihood of leading to infection and further causing serious symptoms, conditions and/or complications (e.g. infants, young children, the elderly, and immunocompromised people). In some settings, for example, without limitation, in institutions such as hospitals, schools, day care centres, military installations, nursing homes, convalescent homes, residential facilities for dependent elderly people, boats and airplanes, a person is determined to be at risk based on time spent near at least one other person who may be infected. In some situations, because respiratory viruses are ubiquitous, generally any individual can be at risk of respiratory virus exposure. The term 'exposure' means an encounter with a respiratory virus (eg through contact with an infected individual or a contaminated area/surface) that allows infection.

C. Biological Sample and Disposable Tissues

1. Collection by Nosing

In a method according to the invention, the biological sample is collected by blowing its nose in a disposable handkerchief. The term 'biological sample', as used here, therefore refers to a speck, namely: the nasal mucus or nasal secretion extracted by blowing one's nose. In most cases, the collection of the biological sample according to a method of the invention is a self-collection (that is to say that the individual collects the sample himself by blowing his nose vigorously). But it is also envisaged that the patient (e.g. an infant or young child) receives help in blowing his nose (e.g. from a parent or caregiver). This may also be the case for people disabled by illness, advanced age or a physical or mental disability who can get help from someone close to them or a carer. Thus, the expressions "collecting a biological sample by blowing one's nose" and "blowing one's nose", as used interchangeably herein, mean both self-collection and assisted collection, provided that the assistance is not issued by a qualified health professional Generally, the collection of the sample is done at the home, at the place of residence or at the place of life of the individual to be tested.

CORRECTED SHEET (RULE 91) ISA/EP - 14 -

The blowing is done in a classic way and as correctly as possible, for example by following the recommendation not to blow both nostrils at the same time but rather to empty one after the other, keeping the head straight, all blowing reasonably and efficiently. In some cases, it may be advisable to wash your hands before blowing your nose or helping someone else to blow their nose.

Specimen collection is preferably done as soon as possible after the onset of symptoms or after a defined time after contact with a person diagnosed with a respiratory virus, the time before collection will be dictated by the type of virus found in the infected person or population.

2. Collection by Spitting

In a method according to the invention, when the individual to be tested has a productive cough, the collection of the biological sample is done by spitting and more precisely by depositing a sputum in a disposable handkerchief. The terms “productive cough” and “congested cough” are used interchangeably herein and refer to a cough, known as a “wet cough”, which is accompanied by abundant production of mucus and phlegm and obstruction of the airways. Thus, the term “biological sample”, as used here, also designates sputum, also called sputum. In some embodiments, the spit is added to the tissue used by the individual to blow their nose. The expression “collecting a biological sample by sputum”, as used herein, means both self-collection and assisted collection, provided that the assistance is not provided by a qualified healthcare professional. As with the collection by blowing, generally, the collection of the sample by sputum is done at the home, place of residence or place of life of the individual to be tested, and preferably as soon as possible after the onset of symptoms. or after a defined time after contact with a person diagnosed with a respiratory virus.

3. Disposable tissues

The handkerchief used to collect the biological sample can be any well-known single-use, disposable or ephemeral paper handkerchief (of the Kleenex® type marketed by Kimberly-Clark Corporation or similar). The size of these handkerchiefs usually ranges from 18 to 22 cm in length and 18 to 22 cm in width. Of course, tissues larger or smaller than those described above are also suitable for the implementation of the collection method herein.

CORRECTED SHEET (RULE 91) ISA/EP invention. Paper handkerchiefs are generally made of recycled paper, wood fiber, and/or cellulose (in particular cellulose wadding), and can be monolayer or multilayer (for example double or triple thickness). They can be white or colored, plain or patterned. In some preferred embodiments, the tissue used to collect the biological sample is white and unpatterned. Disposable paper handkerchiefs can be perfumed (for example with eucalyptus and/or menthol, or any other perfume). In the practice of the present invention, the handkerchief used to collect the biological sample is preferably unscented. Some disposable tissues may also include protective balms enriched with calendula, aloe vera and vitamin E to soothe and protect the nose from irritation. In certain preferred embodiments, the handkerchief used to collect the biological sample does not contain protective balms. Finally, the tissue used to collect the biological sample must not contain antimicrobial agents, such as citric acid, sodium lauryl sulphate or others which are antiseptic and antiviral agents (and which are for example present in Kleenex tissues ® Anti-Viral™ sold in the United States and Canada).

D. Shipping or Transport Container

Once the sample has been collected, the individual to be tested introduces the soiled handkerchief into a container. By 'soiled handkerchief', we mean here the handkerchief in which the individual blew his nose and/or spat, and which therefore contains the biological sample. The container is then closed with a stopper, or any other suitable closure system, to avoid any further contamination. Preferably, the container intended to collect the biological sample is sterile.

The container must be of adequate volume to hold the handkerchief. For example, the container can have a volume of 20 mL, 30 mL or 50 mL. Preferably, the container has a volume of 50 mL. The container is preferably made of plastic (for example: polypropylene (PP), polyethylene (PE), polyethylene terephthalate (PET) or the like). The container must also have an adequate shape to contain the handkerchief. The container may for example have an elongated shape, such as that of a cylindrical or conical tube having a sufficient diameter, for example a diameter of between 2 and 5 cm.

In some embodiments, the container is a centrifuge tube, such as a 50 mL plastic centrifuge tube, such as centrifuge tubes

CORRECTED SHEET (RULE 91) ISA/EP - 16 - Falcon™ 50mL Conical Centrifuge Tubes from Coming™, Easy Reader™ 50mL Polypropylene Centrifuge Tubes from Fisherbrand™, Nunc™ Sterile 50mL Conical Polypropylene Centrifuge Tubes from Thermo Scientific™, and the like .

In other embodiments, the container is a 50 mL plastic needleless syringe, such as BD Medical brand 50 mL Luer-Lock Plastipak syringes, and the like. In this case, after collecting the sample by blowing and/or spitting, the soiled tissue is introduced into the body of the syringe and the syringe is closed with the corresponding plunger to avoid any further contamination.

It is important to note here that the soiled tissue is introduced alone into the container (i.e. in particular in the absence of any transport medium, such as a viral transport solution (VTM)).

E. Sending or Transport to a Laboratory for Biological Analysis

In a method according to the invention, the closed container containing the soiled handkerchief is then transported to the place of analysis. Delivery may be by any appropriate means. For example, the closed container can be inserted into an envelope and sent by standard mail to a biological analysis laboratory. Alternatively, it can be transported by specialized medical transport. During transport to the analysis laboratory, the closed container does not require storage at a temperature below ambient temperature. The term “ambient temperature” denotes a temperature between about 15°C and about 35°C, for example between 20°C and 30°C.

II - Analysis of Viral Nucleic Acids from a Soiled Handkerchief

The present invention also relates to the use of a soiled handkerchief obtained as described previously for the detection of nucleic acids of a respiratory virus, and/or for the diagnosis of a viral respiratory infection in the individual having provided the biological sample by blowing or spitting. The present invention also relates to a method for detecting viral nucleic acids from a soiled handkerchief, and a method for diagnosing a viral respiratory infection in the individual who provided the biological sample by blowing his nose or spitting. Such methods are generally implemented by a biological analysis laboratory, but it is envisaged that they can be carried out by a research laboratory or a laboratory.

CORRECTED SHEET (RULE 91) ISA/EP suitably equipped clinical or hospital laboratory. The terms “biological analysis laboratory”, “medical analysis laboratory”, and “medical biology laboratory” are used here interchangeably and designate a place where various biological fluids of human origin are analyzed under the responsibility of biologists. doctors, who interpret the results in order to participate in the diagnosis and monitoring of certain diseases. In the context of the present invention, the biological analysis laboratory must be equipped to carry out diagnostic tests making it possible to determine the presence or absence of a respiratory virus in a sample, in particular the presence or absence of nucleic acids of this virus under the required conditions of microbiological safety.

More specifically, a method according to the invention for the detection of nucleic acids of a respiratory virus from a soiled handkerchief comprises the following steps consisting in: (a) recovering the biological sample from the soiled handkerchief (the soiled handkerchief having been obtained as described above); (b) detecting the presence of viral nucleic acids in the recovered biological sample and, (c) if viral nucleic acids have been detected, concluding that the respiratory virus is present in the biological sample and possibly diagnosing a respiratory infection d viral origin in the individual who collected the biological sample.

A. Recovery of the Biological Sample Contained in the Soiled Handkerchief

In general, on arrival at the biological analysis laboratory, the container is handled under appropriate biosafety conditions aimed at preventing the accidental risk of personnel exposure to pathogens. A biological analysis laboratory knows how to apply containment measures adapted to the identified risk. These confinement measures are of the architectural type (presence of an airlock, filtration of the extracted air, etc.) and organizational (dedicated equipment for the technical room, storage of protective clothing, cleaning of the premises, etc.. ..).

After receipt, the first step of the method according to the invention consists in recovering the biological sample from the soiled handkerchief. In the context of the present invention, the term "recover" means to isolate, separate or dissociate the biological sample from the soiled tissue. These isolations, separations or dissociations can be carried out by any appropriate method provided that this method does not denature the biological sample.

CORRECTED SHEET (RULE 91) ISA/EP -Io-

In certain embodiments, the biological sample is recovered using an aqueous solution, preferably a physiological solution. As used herein, the term "physiological solution" means any isotonic buffer solution. Examples of suitable saline solutions include, without limitation, saline solutions commonly used in biological or biochemical research such as: saline (composed of distilled water and sodium chloride (NaCl) diluted 9:1000); Ringer's fluid; the Krebs-Henseleit solution; phosphate buffered saline (PBS), Tris buffered saline (TBS); Hank's Balanced Salt Solutions (HBSS); Earle's balanced salt solutions (EBSS); saline solutions of sodium citrate (SSC); HEPES buffered saline solutions (HBS); and Gey's Balanced Salt Solutions (GBSS). In some embodiments, the biological sample is collected using PBS.

The recovery of the biological sample from the soiled handkerchief is done by “washing” or by “wetting” the soiled handkerchief with a physiological solution. The biological sample is fluidized by the physiological solution, and is therefore found in the aqueous phase, which allows a separation of the biological sample (liquid) from the handkerchief (solid). It is possible to carry out a single washing or wetting, but preferably several washings or wettings are carried out successively. Even more preferably, the washings or wettings are carried out with one and the same aliquot of physiological solution used several times. The volume of physiological solution used to recover the biological sample is chosen in such a way that it makes it possible to recover the biological sample with the greatest possible yield while avoiding excessive dilution of the biological sample. A suitable volume of physiological solution (eg PBS) is generally between about 2 and about 8 mL, for example about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, or about 8ml. The washing or wetting step can be carried out in the shipping or transport container where the patient placed the soiled handkerchief. Alternatively, upon arrival at the biological analysis laboratory, the soiled handkerchief is removed from the shipping or transport container and introduced into another container for the biological sample recovery step.

To separate the biological sample extracted from the aqueous phase of the handkerchief, any suitable method can be used. For example, if the container

CORRECTED SHEET (RULE 91) ISA/EP - 19 - used is a centrifuge tube, it is possible to pour the aqueous phase into a new container while keeping the handkerchief in the centrifuge tube. The tissue can also be pressed to the bottom of the tube to optimize the volume of aqueous phase collected. Alternatively, the aqueous phase can be aspirated and introduced into a new container. If the shipping or transport container used is a needleless syringe, the biological sample in the aqueous phase is collected in another container by activating the plunger, which expels the aqueous phase from the body of the syringe. Those skilled in the art are able to modify or adapt these methods depending on the type of container used.

A specific example of biological sample recovery from a soiled tissue contained in a needleless plastic syringe includes the following steps:

(a) Open the syringe by withdrawing the plunger while keeping the soiled tissue at the bottom of the syringe;

(b) Place the syringe over an empty 5-10 mL tube;

(c) Add 6 mL of phosphate-buffered saline (PBS) to the barrel of the syringe to properly soak the handkerchief, and keep the soaked handkerchief in the syringe for 1 minute;

(d) Put the plunger in the syringe to collect, in the empty tube, all the liquid coming out of the syringe;

(e) Remove the plunger from the syringe, and introduce the collected liquid into the barrel of the syringe; and

(f) Repeat steps (d) and (e) twice.

This method makes it possible to recover a final volume of about 2 to about 5 mL of PBS containing the biological sample.

B. Detection of Viraus Nucleic Acids

The next step of the method according to the invention includes the detection of viral nucleic acids in the biological sample recovered from the soiled handkerchief. As used herein, the term "viral nucleic acid detection" broadly refers to a process of determining the presence or absence of viral nucleic acids in the biological sample and/or determining an amount or a level of viral nucleic acids in the biological sample.Thus, the detection can be qualitative, quantitative, or semi-quantitative.

CORRECTED SHEET (RULE 91) ISA/EP Qualitative and semi-quantitative detection include comparison with a predetermined value.

In the context of the present invention, the detection can be carried out using any molecular technique known in the art and making it possible to detect, at least in part, the viral genome (DNA or RNA). “Nucleic Acid Amplification Tests” (NAATs) means a technique that uses the extraction of minute quantities of DNA or RNA and replicates them many times. , making it possible to detect minimal traces of a microorganism in a biological sample, without having to resort to a culture. The implementation of nucleic acid amplification techniques has revolutionized the diagnosis of viral infections of the respiratory tract due to their high sensitivity and specificity, the rapid identification of the virus and the possibility of detecting pathogens that could not not be identified by conventional diagnostic methods (Ginocchio et al., J. Clin. Microbiol., 2011, 49(9): S44-S48; Charlton et al., Clin. Microbiol. Rev., 2018, 32: c00042 -18). In addition, rapid and fully automated commercial NAAT techniques, which allow the detection of a wide panel of respiratory viruses, have been developed. With these "multiplex" techniques, it is possible to analyze a variable number of samples and to apply diagnostic algorithms (Charlton et al., Clin. Microbiol. Rev., 2018, 32: e00042-18) taking into account takes into account parameters such as patient characteristics (immunosuppression, basal respiratory diseases, etc.) and virus seasonality.

Thus, in certain embodiments, the detection of viral nucleic acids in the biological sample recovered from the soiled handkerchief is carried out by a nucleic acid amplification technique. Nucleic acid amplification techniques include, in particular, PCR (Polymerase Chain Reaction), in particular real-time PCR after reverse transcription (RT-PCR); the NASBA technique (nucleic acid sequence-based amplification); the TMA (transcription-mediated amplification) technique; the LAMP (loop-mediated isothermal amplification) technique; the SDA (strand displacement amplification) technique; the LCR (ligase chain reaction) technique, the MLPA (multiplex ligation-dependent amplification) technique and the like. PCR (which quantifies DNA, in real time, during the amplification reaction) and RT-PCR

CORRECTED SHEET (RULE 91) ISA/EP - 21 -

(which quantifies RNA by performing the reverse transcription of message RNA (mRNA) and amplifying the resulting complementary DNA (cDNA)) have nowadays become references in the detection of respiratory viruses. Thus, in certain embodiments, the detection of viral nucleic acids in the biological sample recovered from a soiled tissue is carried out using a PCR or RT-PCR technique.

There are many kits, tests and machines for detecting, by amplification, the nucleic acids of respiratory viruses (Hanson and Couturier, Clin. Infect. Dis., 2016, 63: 1361-1367) which can be used in the practice of a method according to the present invention. Assays can be single (detection of a single respiratory virus), multiplex (simultaneous detection and differentiation of at least two (types or subtypes of) respiratory viruses) or panel (simultaneous detection and differentiation of a wide range respiratory viruses and possibly bacteria responsible for respiratory infections). There are indeed tests on multiplex panel with high complexity, which make it possible to simultaneously and quickly detect up to 20 respiratory viruses (Mahony et al., J. Clin. Microbiol., 2007, 45: 2965-2970), 18 viruses and 2 or 3 atypical bacteria (Gonsalves et al., Methods, 2019, 158: 61-68), 18 viruses and 4 respiratory bacteria (Beckmann and Hirsch, J. Med. Virol., 2016, 88: 1319-1324) and a total of 33 respiratory pathogens (Fast Track Diagnostics, 2018, www.fast-trackdiagnostics.com/human-line/products/ftd-respiratory-pathogens-33).

Examples of suitable respiratory panels that can be used in the practice of the present invention include, without limitation, those marketed by bioMérieux: the BIOFIRE® FILMARRAY® respiratory panel (which allows the simultaneous analysis of 17 viruses and 3 bacteria causing diseases), the BIOFIRE® FILMARRAY® Respiratory Panel 2 (RP2) (which detects 21 pathogens (17 viruses and 4 bacteria) responsible for respiratory diseases), and the BIOFIRE® FILMARRAY® Respiratory Panel 2 Plus (RP2plus) ( which detects 22 pathogens (18 viruses and 4 bacteria) responsible for respiratory infections). Other respiratory panels are marketed by Qiagen, such as the QIAstatDx® Respiratory Panel (which detects 18 viruses and 3 bacteria causing respiratory diseases) and the ResPlex I and II tests; or by Luminex Molecular Diagnostics, such as the xTAG™ Respiratory Viral Panel (R VP) Fast v 2.0 (which allows the detection of 19 types and subtypes

CORRECTED SHEET (RULE 91) ISA/EP respiratory viruses); or by Fast Track Diagnostics, such as for example the Fast Track Diagnostics Respiratory Pathogen 21 and 33 (FTD®-RP21 and FTD®-RP33) (which respectively detect 21 and 33 respiratory pathogens); or by Genomica, such as CLART® PneumoVir (which allows the detection of 17 respiratory viruses); or by Eurogentec, such as the RespiFinder 19 and SMART 22 (which respectively allow the detection of 19 and 22 respiratory pathogens). Other examples of tests and panels that may be used in the practice of the present invention include, without limitation, MultiCode-PLx RVP (EraGen Biosciences, USA), Seeplex® RV, RV-12 and RV-15 (Seegene Inc., Republic of Korea), Magicplex RV Panel Real-Time Test (Seegene Inc., Republic of Korea), Panther Fusion® (Hologic Inc., USA), NGEN™ RVA ASR (Nanogen Inc. ., USA), Pro-FLu+ and Pro-Flu ST (Prodesse GenProbe, USA), Infiniti® RVP Plus (AutoGenomics, USA), Simplexa™ Flu A/B/RSV/FluA /HINl (Focus Diagnostics, United States), the eSensor Respiratory Viral Panel (Gen-Mark Dx, United States), the Easyplex respiratory pathogen 12 kit (Ausdiagnostics, Australia), the Verigene® Respiratory Virus Plus (Nanosphere Inc., United United States) and Allplex™ RV1, RV2 and RV3 panels from Seegene (Republic of Korea).

Kits have even been developed for the detection, by PCR or RT-PCR amplification, of the coronavirus SARS-CoV-2, which is responsible for covid 19. These kits include, without limitation, TaqPath COVID- 19 CE-IVD RT- PCR kit (from Life Technologies Thermofisher Scientific), Allplex™ 2019 nCoV Assay (from Seegene), Cobas SARS-CoV-2 (from Roche Molecular Systems), Standard M nCoV Real-Time Detection kit (from SD Biosensor), VIASURE SARS- CoV-2 Real Time PCR Detection (by Certaint), GeneFinder COVID-19 PLUS RealAmp Kit (by Osang Healthcare), PRESTO 2019-nCoV Direct qPCR kit (by AAZ), Novel Coronavirus (2019-nCoV) Nucleic acid diagnostic kit (PCR Fluorescence probing) (from Sansure Biotech), AmoyDx Novel Coronavirus (2019-nCoV) Detection kit (from Amoy Diagnostics), Novel Coronavirus (2019-nCoV) Nucleic acid detection kit (Fluorescence PCR method) (from Suzhou Tianlong Biotechnology), Bosphorus Novel Coronavirus (2019-nCoV) Detection kit v2 (1 mastermix) (from Anatolia Geneworks), Bosphorus Novel Coronavirus (2019 - nCoV) Detection kit (2 mastermixes) (from Anatolia Geneworks), Vitassay qPCR SARS- CoV-2 (from Vitassay Healthcare), Detection Kit for 2019 Novel Coronavirus (2019- nCoV) RNA (PCR-Fluorescence Probing (from DAAN Gene) , EurobioPlex SARS-CoV-2 Multiplex/real-time RT-PCR (from Eurobio Scientific), Abbott RealTime SARS-

CORRECTED SHEET (RULE 91) ISA/EP CoV-2 (from Abbott), COVID- 19 Nucleic acid detection kit (from Wuhan Healthcare Biotechnology), Quantivirus real-time PCR (SARS-CoV-2) coronavirus detection test (from Diacarta), Novel Coronavirus (2019-nCoV ) Real Time Multiplex RT-PCR Kit (Detection for 3 Genes), (from Liveriver), VitaPCR SARS-CoV-2 Assay (from Credo Biomedical/Trenton Biomedical), LabGun™ COVID-19 Assay (from Labgenomics), QIASTAT-DX Respiratory SARS-CoV-2 panel (from Qiagen), COVID- 19 Coronavirus Real Time PCR kit (from Jiangsu Bioperfectus Technologies), PerkinElmer® SARS-CoV-2 Real-time RT-PCR Assay (from Perkin Elmer), VIRELLA SARS- CoV-2 seqc real time RT-PCR kit (from Gerbion), Novel Coronavirus (2019-nCoV) RT-PCR Detection Kit - Fosun 2019-nCoV qPCR (from Shanghai Fosun Long March Medical Science), Xpert® Xpress SARS-CoV- 2 or SARS-CoV-2/Flu/RSV (from Cepheid) and Multiple Real-Time PCR Kit Detection 2019-nCoV (from XABT Beijing Applied Biological Technologies).

Before analysis by amplification, it is necessary to extract the viral nucleic acids from the recovered biological sample. This extraction requires cell lysis, inactivation of cellular nucleases, and separation of the desired nucleic acid from cellular debris and proteins. Methods for extracting viral nucleic acids are known in the art. Many kits can be used for the extraction of viral DNA or RNA from human body fluids or tissues and are commercially available, for example, from ThermoFischer Scientific (USA), Qiagen ( Germany), Macherey-Nagel (Germany), Roche (Switzerland), Promega (United States), PerkinElmer (United States), MGI (China). User guides, which describe in detail the protocol to be followed, are generally included in all these kits. Sensitivity, processing time and cost may vary from kit to kit. Medical biologists in analytical laboratories know how to select the most appropriate kit(s) for a particular situation. Additionally, amplification assay kits sometimes contain the reagents needed to extract viral nucleic acids.

A method, according to the present invention, for the detection of nucleic acids of a respiratory virus from a soiled tissue obtained as described herein, can provide at least one of the following results: presence or absence of a virus respiratory virus in the biological sample tested, identity of the respiratory virus present in the sample tested, and amount or level of respiratory virus present in the sample

CORRECTED SHEET (RULE 91) ISA/EP tested. The quantity of respiratory virus can for example correspond to a viral load or a viral titer. The terms “viral load” and “viral titer” are used interchangeably herein. They designate a numerical expression of the quantity of virus relative to the number of cells collected on the handkerchief. This quantity of cells roughly reflects the quality of the blowing or spitting deposited on the handkerchief.

C. Presence of a Respiratory Virus in the Biological Sample and Diagnosis of a Respiratory Viral Infection

In a method according to the invention, if viral nucleic acids have been detected during the analysis of the biological sample by an amplification technique, it is concluded that a respiratory virus is present in the biological sample recovered. soiled handkerchief. In some embodiments, the method further includes diagnosing a viral respiratory infection in the individual who collected the biological sample. As used herein, the term “diagnosis of a viral respiratory infection” means a conclusion based on the identification of the respiratory virus and possibly the determination of the viral load (or any other numerical expression of the amount or level virus in the biological sample). The quantity of cells obtained on the handkerchief will allow, if necessary, to certify the successful completion of the self-sampling. However, the results of a method according to the invention must be interpreted in the context of all relevant clinical data. Thus, it is up to the practitioner to make the definitive medical diagnosis.

The results of a method according to the invention can make it possible to administer an adequate treatment to the individual who provided the biological sample by blowing his nose and/or spitting and/or to impose a quarantine or an isolation on this individual and /or initiate identification of contact cases, depending on the nature of the respiratory virus. The term “treatment” is used herein to characterize a method or process that aims (1) to slow or stop the progression or worsening of viral respiratory infection, and/or (2) to improve at least one symptom of viral respiratory infection (eg, by reducing the severity or duration of the symptom), and/or (3) preventing at least one symptom of the viral respiratory infection, and/or (4) suppressing the respiratory infection infection and/or (5) to limit the transmission of the respiratory infection to those around As used herein, the terms “quarantine” and “isolation” refer to a

CORRECTED SHEET (RULE 91) ISA/EP - 25 - barrier measure which consists of isolating a person for a certain period of time, in the event of a diagnosis of viral respiratory infection, to prevent the contagion of third parties and the spread of the virus.

In addition, the methods provided herein have a variety of other uses, including, without limitation, (1) in the detection of individuals who have been exposed to a respiratory virus and who have an impending but non-symptomatic infection (e.g. in the event of an epidemic); (2) in monitoring the response to a vaccine or drug, either as part of a clinical trial or to monitor population immunity; and (3) in screening for impending viral respiratory infection prior to deployment (e.g., military or civilian deployment such as boarding a cruise ship). for example between 20°C and 30°C.

III - Biological Sample Collection Kits for the Detection of Respiratory Viruses

In another aspect, the present invention provides kits comprising material useful in the implementation of a method for collecting at least one biological sample according to the invention.

Thus, for example, a kit according to the invention may comprise a container intended to collect a soiled handkerchief, and an element for identifying the sample. The container, for example a centrifuge tube or a needleless syringe as described above, may be contained in a plastic packaging pouch, to be removed before use. Here, “identification element” means any means allowing the sample to be linked, without any ambiguity, to the patient who provided this sample. In some embodiments, the identification element makes it possible to enter, for example in a handwritten manner, the information concerning the identity of the patient who provided the biological sample (for example: surname, first name, date of birth and sex) . In other embodiments, the identification element may comprise a unique identification number or a barcode specifically assigned to the patient who has previously provided his identity or who provides his identity on a sheet or a form, which is also included in the kit and has this same unique identification number or barcode. This identification element can be in any appropriate form, for example in the form of a self-adhesive label, which can be stuck on the container before dispatch, or in the form of a

CORRECTED SHEET (RULE 91) ISA/EP - 26 - card or a form to be inserted in the envelope before sending. The identification element or another piece of information can make it possible to provide other useful information such as, for example, the date and time of the collection of the biological sample, the address for sending the results, the name of the attending physician, the patient's state of health, the drug treatment(s) followed by the patient, etc.

In certain embodiments, a kit according to the invention further comprises a handkerchief contained in an individual plastic bag.

A kit according to the invention can also comprise an envelope for sending or transporting to an analysis laboratory.

A kit according to the present invention may also include an instruction manual for the use of the kit according to a sample collection method described here and/or a list of addresses of appropriate biological analysis laboratories.

A kit according to the invention generally allows the collection of a single biological sample from a single individual. However, it is envisaged that a kit contains enough containers and identification elements to allow the collection of several biological samples from several individuals (for example for a family, or for a community).

A kit according to the invention may optionally contain a notice or notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceutical or biological products, such notice reflecting the approval, by the agency, manufacture, use or sale for human use.

The individual components of the kit are preferably kept in close containment for commercial sale.

An identifier, for example, barcode, radio frequency, etc., may be present in or on the kit. The identifier can be used, for example, to uniquely identify the kit for the purposes of quality control, inventory control, tracking movement between workstations, etc.

Other aspects and advantages of the present invention are described in the following figures and examples, which should be considered as illustrative and not limiting the scope of the invention.

CORRECTED SHEET (RULE 91) ISA/EP - 27 -

Examples

The following examples describe some embodiments of the present invention. However, it is understood that the examples and figures are presented for illustrative purposes only and in no way limit the scope of the invention.

Experiences

Several experiments were carried out. They made it possible to validate the approach to the analysis of handkerchiefs for the detection of respiratory viruses. Validation was performed for three different situations:

1. The detection of respiratory viruses after collecting handkerchiefs in a crèche over the course of a year;

2. Detection of respiratory viruses after collection of tissues in a nursery school; and

3. Individual detections and diagnoses in symptomatic individuals.

1. Detection of Respiratory Viruses After Collecting Handkerchiefs in a Nursery for a Year

An operation was carried out in a crèche welcoming children from September 2018 to September 2019. The children were structurally separated into "little ones" (<18 months) and "big ones" (>18 months) and welcomed in independent rooms in the within the same nursery. Each week, the handkerchiefs of all the children were collected in a group and not individually in order to detect, according to the present invention, the viruses which could circulate over time within the reception structure.

Figure 1 summarizes the viruses detected throughout the year (each week is identified as "S") for the "little ones" (room B) and for the "big ones" (room G) within the crèche. Each box that has been checked indicates that a virus has been detected during the week in question. Significantly, we note the independent circulation of seasonal viruses according to the room considered. In other words, the "little ones" can be infected by certain viruses at certain times, without the "big ones" being infected by the same virus at the same time. It should also be noted that, in addition to seasonal respiratory viruses, other viruses responsible for childhood illnesses were searched for on handkerchiefs (B 19: parvovirus B 19; VZV: varicella and shingles virus; CMV: cytomegalovirus).

This study demonstrates the feasibility of the virus detection approach on tissues collected weekly. She gave an objective view of

RECTIFIED SHEET (RULE 91) ISA/EP - 28 - viral ecology over a full year in a crèche receiving two child populations of different ages. This study also reveals that the viruses thus seem to circulate differently from the moment the children are physically separated in different rooms and the rules of hygiene are respected.

The practical consequences were to inform the parents of the circulation of certain viruses throughout the year and thus to explain certain symptoms which they could observe on their children, or on themselves, while reassuring them on the etiology of these generally benign clinical manifestations. The generalization of this approach could ultimately lead to less use of antibiotics since the identification of a virus provides a major argument for the non-use of antibiotics, which are ineffective on viruses.

2. Detection of Respiratory Viruses After Collection of Tissues in a Nursery School

In a second study, a fairly similar protocol was carried out by collecting used tissues in two kindergarten classes in small and large sections. Each week, handkerchiefs used by schoolchildren were collected and analyzed for the presence of respiratory viruses according to the protocol of the present invention.

Figure 2 presents a table that indicates the viruses detected each week of the school year (2019-2020). This school year having been marked by confinement, this abruptly stopped the study at the beginning of March. Each box that has been checked indicates the detection of the virus in the week in question.

This second study made it possible to validate the experimental approach and demonstrates the possibility of detecting viruses on handkerchiefs according to the protocol of the present invention. Again, an often dissociated circulation of viruses was observed between the two groups of schoolchildren. This is to be linked to the phenomena of immunization throughout life. Young students infected with a virus in the early years become less infected with these same viruses in subsequent years.

3. Individual Detections and Diagnoses in Symptomatic Persons

Another study was conducted with the aim of comparing the reliability of screening for SARS-CoV-2 according to the method of the present invention and that of the reference method. The latter is defined by the state of the art and the recommendations of the HAS, namely a nasopharyngeal swab for the diagnosis of COVID-19.

CORRECTED SHEET (RULE 91) ISA/EP It was proposed to 17 adults presenting for a diagnosis of COVID and benefiting from a nasopharyngeal swab according to the state of the art, to blow their nose according to the protocol described here. The samples analyzed according to the classic procedure for detecting SARS-CoV-2 in the laboratory on nasopharyngeal swabs revealed 9 positive cases. The handkerchiefs of all these people (n=17) were analyzed according to the procedure described here and 6 were found to be positive. It should be noted that the 6 samples presenting a viral load compatible with a risk of transmission of the virus (i.e. an RT-PCR signal <30 Ct) were all detected positive on the handkerchiefs. No false positive signals were noted after analysis of the tissues on the negative nasopharyngeal samples.

Figure 3 shows the Ct (threshold cycle) values obtained on the SARS-CoV-2 N gene depending on whether the detection technique was performed on the nasopharyngeal swab (NPS) or on the tissue collected from the same nobody. The three individuals with the NPS having the highest Cts (30, 31 and 36, i.e. lower viral loads) were not detected during the analysis of the tissues.

These results are in line with what was expected, namely a lower sensitivity for the detection of SARS-CoV-2 on tissues, compared to the nasopharyngeal swab. Several recent studies working on different matrices than those recommended, in particular saliva or oropharyngeal swabs, indicate comparable results with less sensitivity than the standard method (nasopharyngeal swab). Current discussions focus on the fact that a less sensitive technique would nevertheless be the best reflection of an individual's risk of contagiousness. An analysis of the handkerchiefs would therefore make it possible to correctly identify people at risk of transmitting SARS-CoV-2.

Work has also been carried out from time to time on handkerchiefs sent to the laboratory and analyzed free of charge and experimentally for people with clinical symptoms suggestive of a viral respiratory infection. The tissues were sent by post or directly to the laboratory by the people themselves in a plastic bag. The analysis of these tissues according to the protocol described here made it possible to identify influenza B viruses, metapneumoviruses, rhinoviruses or SARS-CoV-2. These one-off experiments indicate the feasibility of analyzing handkerchiefs on an individual basis, even at a distance from the blowing, and of establishing an etiological diagnosis explaining the symptoms experienced.

CORRECTED SHEET (RULE 91) ISA/EP - 30 -

Conclusion

All of the studies carried out indicate that the collected sniffles are good matrices for the detection of respiratory or respiratory-transmitted viruses. The analysis of used tissues provides information on the viruses circulating during a given period and in a defined population. Knowing that there are only very few antiviral treatments that are effective on these seasonal viruses, analyzing the tissues and providing information on the circulating viruses makes it possible to inform the population and avoid the unnecessary use of antibiotics while strengthening the implementation of barrier measures to prevent viral dissemination. At the individual level, the analysis of handkerchiefs makes it possible to make a diagnosis in people presenting symptoms suggestive of a viral infection. The clinical sensitivity of the detection of viral genomes on handkerchiefs is not fully established compared to the state of the art based on a nasopharyngeal swab, but a positive test is a sure sign of an ongoing infection. The advantage of handkerchief screening is the possibility of carrying out a self-sample, thus not exposing a possible sampler and simply being able to send the handkerchief without any special storage precautions to a laboratory authorized for the detection of viral genomes.

CORRECTED SHEET (RULE 91) ISA/EP

Claims

Claims

1. Method for detecting nucleic acids of a human respiratory virus from a handkerchief containing a biological sample from an individual to be tested, the method comprising the following steps: obtaining a handkerchief containing a biological sample, where the biological sample was collected by a method comprising the following steps in which: the individual to be tested blows his nose in a single-use handkerchief; the individual introduces the soiled handkerchief alone into a container and closes the container; and the container containing the soiled handkerchief is taken to a biological analysis laboratory; recover the biological sample contained in the soiled handkerchief using a physiological solution, in particular a saline phosphate buffer; detect the presence of viral nucleic acids in the recovered biological sample; and if viral nucleic acids have been detected, conclude that the respiratory virus is present in the biological sample and possibly diagnose a viral respiratory infection in the individual who collected the biological sample.

2. Method according to claim 1, characterized in that the method for collecting the biological sample further comprises a step in which: the individual to be tested spits into the disposable handkerchief.

3. Method according to claim 1 or claim 2, characterized in that the container has a volume of 50 ml.

4. Method according to any one of claims 1 to 3, characterized in that the container is a plastic centrifuge tube or a plastic needleless syringe. Method according to any one of Claims 1 to 4, characterized in that the soiled handkerchief is transported to a biological analysis laboratory at ambient temperature. Method according to any one of Claims 1 to 5, characterized in that the container containing the soiled handkerchief is inserted into an envelope and transported to a biological analysis laboratory by post or by specialized medical transport. Method according to any one of Claims 1 to 6, characterized in that the respiratory virus is chosen from the group consisting of influenza viruses, parainfluenza viruses, respiratory syncytial virus (hRSV), metapneumoviruses (hPMV), adenoviruses , rhinoviruses, and coronaviruses. Method according to any one of Claims 1 to 6, characterized in that the respiratory virus is chosen from the group consisting of influenza A viruses, in particular the subtypes H1N1, H3N2, A(H5N1), A( H7N7), A(H9N2), and A(H7N9), influenza B viruses, parainfluenza viruses type 1, type 2, type 3 and type 4, respiratory syncytial virus (hRSV) of the sub- group A, respiratory syncytial virus (hRSV) subgroup B, metapneumoviruses subtype A, metapneumoviruses subtype B, adenoviruses responsible for respiratory infections, and coronaviruses 229E, NL63, OC43 , HKU1, B814, SARS-CoV, MERS-CoV, and SARS-CoV-2. Method according to any one of Claims 1 to 8, characterized in that the individual to be tested is suspected of being infected with a respiratory virus or is at risk of being infected with a respiratory virus. Method according to any one of Claims 1 to 9, characterized in that the individual to be tested is not a pediatric patient. Method according to any one of Claims 1 to 10, characterized in that detecting the presence of viral nucleic acids in the recovered biological sample is carried out by a nucleic acid amplification technique, in particular a technique chosen from the techniques of PCR, RT-PCR, NASBA, TMA, LAMP, SDA, LCR and MLPA. Method for collecting a biological sample for the detection of a human respiratory virus in an individual to be tested, the method comprising the following steps in which: the individual to be tested blows his nose and/or spits into a single-use handkerchief; the individual introduces the soiled handkerchief into a container and closes the container; and the container containing the soiled handkerchief is taken to a biological analysis laboratory. Method for collecting a biological sample according to claim 12, characterized in that the respiratory virus is chosen from the group consisting of influenza viruses, parainfluenza viruses, respiratory syncytial virus (hRSV), metapneumovirus (hMPV), adenoviruses, rhinoviruses, and coronaviruses. Method for collecting a biological sample according to claim 12 or claim 13, characterized in that the respiratory virus is chosen from the group consisting of influenza A viruses, in particular the subtypes H1N1, H3N2, A(H5N1) , A(H7N7), A(H9N2), and A(H7N9), influenza B viruses, parainfluenza viruses type 1, type 2, type 3 and type 4, respiratory syncytial virus (hRSV) of subgroup A, respiratory syncytial virus (hRSV) of subgroup B, metapneumoviruses of subtype A, metapneumoviruses of subtype B, adenoviruses responsible for respiratory infections, and coronaviruses 229E, NL63, OC43, HKU1, B814, SARS-CoV, MERS-CoV, and SARS-CoV-2. Method for collecting a biological sample according to any one of claims 12 to 14, characterized in that the individual to be tested is suspected of being infected with a respiratory virus or is at risk of being infected with a respiratory virus . Method for collecting a biological sample according to any one of Claims 12 to 15, characterized in that the individual to be tested is not a pediatric patient. Method for collecting a biological sample according to any one of Claims 12 to 16, characterized in that the soiled handkerchief is introduced alone into the container. Method for collecting a biological sample according to any one of Claims 12 to 17, characterized in that the container has a volume of 50 mL. A method of collecting a biological sample according to any one of claims 12 to 18, characterized in that the container is a plastic centrifuge tube or a plastic needleless syringe. Method for collecting a biological sample according to any one of Claims 12 to 19, characterized in that the container containing the soiled handkerchief is transported to a biological analysis laboratory at ambient temperature. Method for collecting a biological sample according to any one of claims 12 to 20, characterized in that the container containing the soiled handkerchief is inserted into an envelope and transported to a biological analysis laboratory by post or by specialized medical transport . Use of a biological sample obtained by a collection method as defined in any one of claims 12 to 21, for the detection of nucleic acids of a respiratory virus and/or for the diagnosis of a viral respiratory infection in the individual who provided the biological sample. A method for detecting nucleic acids of a respiratory virus from a soiled handkerchief, the method comprising the following steps: recovering the biological sample contained in a soiled handkerchief, the soiled handkerchief having been obtained by a method for collecting a biological sample as defined in any one of claims 12 to 21; detecting the presence of viral nucleic acids in the recovered biological sample; and if viral nucleic acids have been detected, concluding that the respiratory virus is present in the biological sample and possibly diagnosing a viral respiratory infection in the individual who collected the biological sample. Method for detecting nucleic acids of a respiratory virus from a soiled handkerchief according to claim 23, characterized in that the biological sample is recovered from the soiled handkerchief using a physiological solution, in particular a phosphate buffered saline. Method for detecting nucleic acids of a respiratory virus from a soiled handkerchief according to claim 23 or claim 24, characterized in that detecting the presence of viral nucleic acids in the recovered biological sample is carried out by a technique for amplification of nucleic acids, in particular a technique chosen from the techniques of PCR, RT-PCR, NASBA, TMA, LAMP, SDA, LCR and MLPA. Kit for collecting a biological sample by blowing and/or spitting for the detection of a respiratory virus in an individual to be tested, the kit comprising: a container intended to collect a handkerchief soiled by blowing and/or spitting from the individual to test ; and an element for identifying the biological sample. Kit for collecting a biological sample according to claim 26, characterized in that the container has a volume of 50 mL. A biological sample collection kit according to claim 26 or claim 27, characterized in that the container is a plastic centrifuge tube or a plastic needleless syringe. Kit for collecting a biological sample according to any one of Claims 26 to 28, characterized in that the kit also comprises at least one component chosen from: a tissue paper contained in an individual plastic bag, a mailing envelope or transport to a biological analysis laboratory, an instruction manual for the kit according to a method according to any one of claims 12 to 21, and a list of addresses of appropriate biological analysis laboratories.