EP4373977A1

EP4373977A1 - Use of torque teno virus (ttv) as a marker to determine the risk of complications in a patient admitted to a healthcare facility

Info

Publication number: EP4373977A1
Application number: EP22754486.3A
Authority: EP
Inventors: François Mallet
Original assignee: Biomerieux SA; Bioaster
Current assignee: Biomerieux SA; Bioaster
Priority date: 2021-07-19
Filing date: 2022-07-19
Publication date: 2024-05-29
Also published as: FR3125300A1; CN117957333A; WO2023002120A1

Abstract

The present invention relates to a method for in vitro or ex vivo assessment of the risk of complications in a patient admitted to a healthcare facility, the method comprising measuring the viral load of at least one torque teno virus (TTV) in a biological sample of said patient, characterised in that said patient is not undergoing immunosuppressive treatment.

Description

DESCRIPTION

TITLE: USE OF THE TORQUE TENO VIRUS (TTV) AS MARKERS TO DETERMINE THE RISK OF COMPLICATION IN A PATIENT ADMITTED TO A HEALTHCARE ESTABLISHMENT

INTRODUCTION

Secondary infections (or healthcare-associated infections - IAS) are major complications of medical care, particularly in medical care structures such as hospitals where we will speak of nosocomial infections. Nosocomial infections occur in 20 to 40% of patients admitted to the intensive care unit and result in lengthening of hospital care often associated with the use of invasive medical devices, as well as the greater administration of antibiotics. In the long term, morbidity and mortality are also higher.

The occurrence of healthcare-associated infections has been particularly exacerbated in recent years, due to the increase in multi-resistant pathogens. The World Health Organization (WHO) estimates the number of nosocomial infections in hospitals in Europe at around 5 million, leading to around 50,000 deaths and an additional annual cost of 13 to 24 billion euros. Recommendations have been published, and the establishment of infection control programs has been encouraged, in particular by the U. S. Department of Health and Human Services, the European Center for Disease Prevention and Control, WHO and the national agencies, for which the prevention and reduction of healthcare-associated infections have become a major priority. According to some models, a test that would reduce the time to identify patients at high risk of contracting healthcare-associated infections would reduce mortality in these patients, with a good cost-effectiveness ratio.

It is therefore important to be able to identify early markers associated with a risk of IAS and, more generally, with a risk of developing a complication during medical treatment, in particular in order to provide an appropriate therapeutic response. as soon as a risk is detected.

Many factors influence the occurrence and development of healthcare-associated infections, such as factors related to patient management but also the patient's general state of health. Several studies have demonstrated the existence of a relationship between immune alterations and an increased incidence of secondary infections (reviewed in (Delano et al., 2016), but these results remain controversial and a more recent study calls into question the existence of a link (Venet et al., 2021).

Some studies have also attempted to establish a link between the phenomenon of viral reactivation (herpesvirus (Hpv) and torque teno virus (TTV) in particular) and the occurrence of treatment-related complications. The phenomenon of viral reactivation exists in particular in non-immunocompromised, seriously ill (sepsis) or injured (trauma, burn, surgery) patients who present with immune paralysis (or immunoparalysis, characterized by a dysfunction of the adaptive and innate immune systems, in response to an initial hyper-inflammatory state).

To date, the clinical implications of viral reactivation are not clearly established. In particular, it is not clear whether the viruses that reactivate should be considered as simple markers reflecting an alteration of the immune system, or on the contrary, as pathogenic agents favoring secondary infections and requiring the establishment of a preventive treatment (Limaye et al, 2010).

Recently, Mallet et al. (Mallet et al., 2019 - Intensive Care Medicine Exp. 7:28) published the results of a study suggesting the existence of a link between the co-reactivation of EBV viruses (virus of Epstein-Barr) and TTV, and a nonsignificant decrease in mortality.

Another recent study, conducted in patients who had undergone kidney transplantation, established that a measurement of viral load on TTV could identify patients at low risk of secondary infection (Strassl et al., The Journal of Infectious Diseases, 2018;218:1191-9). However, the scope of this study remains limited, especially since the patients involved have all received immunosuppressive treatment and are therefore at greater risk of developing infectious diseases. This therefore does not make it possible to establish a credible link between the viral load in TTV and the risk of a secondary infection (or healthcare-associated infection), and therefore to identify which patients are at high risk of developing a secondary infection. . There is therefore still a need for a simple and reliable diagnostic method to identify patients at risk of developing a complication, in particular a healthcare-associated infection, during their medical care within a healthcare facility. .

SUMMARY OF THE INVENTION

A first object of the present invention relates to a method for in vitro or ex vivo evaluation of the risk of complication in a patient admitted to a healthcare establishment, comprising measuring the viral load of at least one torque teno virus (TTV) in a biological sample from said patient, characterized in that said patient is not under immunosuppressive treatment.

According to a preferred embodiment, said method is characterized in that the patient is a patient admitted to a hospital, preferably to the emergency department, the resuscitation department, to an intensive care unit or to a ongoing care.

According to another preferred embodiment, said method is characterized in that said patient is a patient in a septic state, more particularly, in septic shock, a patient suffering from burns, more particularly severe burns, a patient suffering from trauma, more particularly severe trauma, or a patient who has undergone surgery, more particularly major surgery.

According to another preferred embodiment, said method is characterized in that the biological sample comes from a patient in a septic state, suffering from burns, suffering from trauma or having undergone a surgical operation, and admitted to an intensive care unit .

According to another preferred embodiment, said method is characterized in that the complication consists of the occurrence of a healthcare-associated infection (HAI).

According to another preferred embodiment, said method is characterized in that it comprises the implementation of the steps consisting in: a) measuring the viral load of at least one TTV in a biological sample from said patient, b) comparing the viral load determined for said biological sample with a predetermined reference value, and c) establishing a risk of complication when the viral load determined for said sample is higher or lower than the predetermined reference value.

According to another preferred embodiment, said method is characterized in that the predetermined reference value is 10,000 copies/ml.

According to another preferred embodiment, said method is characterized in that it comprises the implementation of the steps consisting in: a) measuring a first viral load of at least one TTV in said biological sample of the patient from a first sample taken at time T1, b) measuring a second viral load of at least one TTV in said biological sample of the patient from a second sample taken at time T2, c) calculating the variation between the viral load at T2 and the viral load at T1, giving an ATTV value, d) establishing a conclusion as to the risk of complication, based on the result of the comparisons.

According to a preferred embodiment, the method is characterized in that it comprises the implementation of the steps consisting in: a) measuring a first viral load of at least one TTV in said biological sample of the patient from a first sample taken at time T1, b) measuring a second viral load of at least one TTV in said biological sample of the patient from a second sample taken at time T2, c) calculating the variation between the viral load at T2 and the load viral at T1 , giving an ATTV value, d) draw a conclusion as to the absence of an increased risk of complication when the ATTV value is within the range from -1.25 to +1.25, preferably -1.20 to +1.20, and more preferably from -1.10 to +1.10. According to another preferred embodiment, said method is characterized in that it also comprises the detection of the presence of at least one Herpesvirus (HPV).

According to another preferred embodiment, said method is characterized in that it also comprises measuring the viral load of at least one Herpesvirus (HPV).

According to another preferred embodiment, said at least one Herpesvirus (HPV) is selected from the group consisting of: CMV, EBV, HHV6 and HSV-1, and is preferably EBV.

According to another preferred embodiment, said method is characterized in that the viral load is measured by amplification, sequencing or hybridization of at least one sequence of TTV, and of at least one Herpes virus where appropriate, preferably by amplification , more preferably by real-time PCR.

According to another preferred embodiment, said method is characterized in that the biological sample is a biological fluid from the patient, said fluid being selected from the group consisting of: blood or its derivatives such as plasma and/or serum , cerebrospinal fluid, urine, and bronchoalveolar lavages.

DETAILED DESCRIPTION

The subject of the present invention is a method for evaluating a risk of complication, for example a healthcare-associated infection (HAI), in a patient not taking immunosuppressive treatment, admitted to a healthcare establishment.

Indeed, the inventors have shown that the torque teno virus (TTV) viral load is an indicator of the risk of complications in patients not taking immunosuppressive treatment, admitted to a healthcare establishment and more particularly in patients treated in an intensive care unit.

In particular, it appears surprisingly that the increase in viral load in TTV after the first week following admission to intensive care is significantly correlated with a longer length of stay in this department. Moreover, both the increase and the decrease in the viral load in TTV are significantly associated with the occurrence of HAI during the first month following admission.

Finally, the co-detection from the first week following the patient's admission, particularly in the intensive care unit, of a high viral load in TTV associated with the presence of at least one herpesvirus, is correlated with a risk of occurrence of a healthcare-associated infection (HAI). It is thus possible to assess early the risk of occurrence of a complication, and in particular the risk of occurrence of HAI, in patients not undergoing immunosuppressive treatment. In the present description, the embodiments can be taken alone or suitably combined by the person skilled in the art.

Methods for assessing the risk of complications in a patient

The subject of the present description is a method for in vitro or ex vivo evaluation of the risk of complications in a patient not taking immunosuppressive treatment, admitted to a health establishment, based on a measurement of the viral load of at least one TTV in a biological sample from said patient. According to a first aspect, a method is described here for the in vitro or ex vivo evaluation of the risk of complication in a patient admitted to a health establishment, comprising measuring the viral load of at least one TTV in a biological sample from said patient, characterized in that said patient is not under immunosuppressive treatment. Surprisingly, by studying the phenomena of viral reactivation in patients in a healthcare establishment, the inventors have identified in patients who are not on immunosuppressive treatment the existence of a correlation between the evolution of the viral load in TTV during his stay in the health establishment and the occurrence of complications, i.e., the unfavorable evolution of his condition, namely in particular the occurrence of infections associated with the care.

According to a preferred embodiment, a variation in the viral load in TTV during the first month following admission to the healthcare establishment indicates a risk of developing an HAI. The expression "assessment of the risk of complication in a patient" or "determine a risk of complication in a patient" means here the determination of a probability for a given subject or patient to develop a complication, as defined below, in the future.

The methods disclosed here represent tools for assessing said risk, and can be combined with other methods, methods or indicators such as clinical examination, taking a biological sample from said patient and detecting one or more biomarkers and/or of one or more infectious agents, in particular bacteria, viruses, parasites or yeasts and filamentous fungi.

By “complication” is meant here the unfavorable evolution of a disease, a state of health or a medical treatment. A "complication", as it is understood here, can be in particular the unfavorable evolution of a disease which may require the installation of one or more invasive medical devices, such as for example a tracheal intubation, the placement of a urinary catheter, the placement of a catheter or a central line.

The term "complication" also means the unfavorable evolution of a disease which may have the effect of extending the duration of care in the said health establishment, in particular in the intensive care unit.

“Complication” also means the death of said patient.

Preferably, the term "complication" also means the unfavorable evolution of a disease that may require the implementation of a specific medical treatment. An example of such a complication is the development of a secondary infection or opportunistic infection, in particular a "care-associated infection" or "HAI", such as an infection by one or more of the infectious agents, for example of bacteria, viruses, parasites or yeasts and filamentous fungi.

In the context of the present description, an infection is said to be "associated with care", if it occurs during the management (diagnostic, therapeutic, palliative, preventive, educational or operative) of a patient by a healthcare professional. health, and if she was not present at the start of treatment. Healthcare-associated infections include infections contracted in a health facility (known as nosocomial infections) but also during care delivered outside this setting. When the infectious state at the start of treatment is not precisely known, a delay of at least 48 hours or a delay longer than the incubation period is commonly accepted to define an HAI.

For surgical site infections, infections occurring within 30 days of surgery or, if an implant, prosthesis or prosthetic material are placed are usually considered to be healthcare-associated. in the year following the intervention. Healthcare-related infection can be of bacterial origin, such as bacteria such as Escherichia coli, Staphylococcus aureus (staphylococcus aureus) or Pseudomonas aeruginosa. The infection can also be of fungal origin, in particular by a yeast of the Candida genus, or a filamentous fungus of the Aspergillus genus, in particular Aspergillus fumigatus. The infection can also be of the viral type, in particular by a virus of the Adenovirus, Herpesvirus, Enterovirus, Rotavirus or HIV type. It can also be the reactivation of potentially pathogenic latent viruses, such as herpes viruses, for example the CMV, EBV, HHV6 and HSV-1 viruses. According to a preferred embodiment, the description relates to an in vitro or ex vivo method for evaluating a risk of complication in a patient not taking immunosuppressive treatment, said complication being the occurrence of a healthcare-associated infection (HAI).

In particular, G IAS can be a nosocomial infection. In this case, the method described here makes it possible to determine the risk of occurrence of a nosocomial infection in said patient. In the case of a patient in a septic state, who is therefore already suffering from a first infection, the process described here makes it possible to determine the risk of occurrence of a secondary infection. The method described here is based in particular on measuring the viral load of at least one TTV in a biological sample from the patient.

“Biological sample” means any sample that can be taken from a subject.

In general, the biological sample must allow the determination of the TTV and/or Herpesvirus (HPV) load. The biological sample, as understood herein, includes, but is not limited to, whole blood, serum, plasma, sputum, nasopharyngeal swabs, urine, stool, skin, cerebrospinal fluid, saliva, gastric secretions, semen, seminal fluid, tears, tissue or spinal fluid, cerebral fluid, trigeminal ganglion sample, sacral ganglion sample, adipose tissue, lymphoid tissue, placental tissue, upper reproductive tract tissue, gastrointestinal tract tissue, genital tissue and central nervous system tissue.

In particular, this sample can be a biological fluid, such as a blood sample or a sample derived from blood, which can in particular be chosen from whole blood (as collected from the venous route, that is to say containing the white and red cells, platelets and plasma), plasma, serum, as well as any type(s) of cells extracted from blood, such as peripheral blood mononuclear cells (or PBMC, containing lymphocytes (B, T and NK cells), dendritic cells and monocytes), subpopulations of B cells, purified monocytes, or neutrophils. The sample to be tested can be used directly from the biological source or following a pre-treatment to modify the character of the sample. For example, such pretreatment may include the preparation of plasma from blood, the dilution of viscous fluids, and so on.

Pretreatment processes may also involve filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, addition of reagents, lysis, etc. Additionally, it may be beneficial to modify a solid test sample to form a liquid medium or to release the analyte.

Preferably, the biological sample is a biological fluid from the patient, said fluid being selected from the group consisting of: blood or its derivatives such as plasma and/or serum, cerebrospinal fluid, urine, and bronchoalveolar lavages. Even more preferably, the biological sample is a sample of blood or one of its derivatives such as plasma and/or serum.

The term "subject" herein refers to a vertebrate, preferably a mammal and most preferably a human. A human subject may also be called a “patient”.

By "patient", we refer here more particularly to a human subject who has come into contact with a health professional, such as a doctor (for example, a general practitioner) or a medical structure or a health establishment (for example, a hospital, and more particularly the emergency department, the intensive care unit, an intensive care unit or a continuing care unit, or a medical structure for the elderly, of the EHPAD type). According to certain embodiments, the patient may be an elderly person, within the framework of a vaccination protocol (in particular in an EHPAD or even at a general practitioner).

As described herein, said evaluation method makes it possible to determine the risk of developing a complication in a patient who is not under immunosuppressive treatment.

“Patient who is not on immunosuppressive treatment” means that said patient is not taking immunosuppressive drugs, or drugs with immunosuppressive side effects, nor is he undergoing any treatment that may induce immunosuppression.

By “immunosuppressive treatment” or “inducing immunosuppression” is meant treatments, in particular drugs, having the effect of reducing, inhibiting or preventing the activity of the immune system. By way of non-limiting example, an "immunosuppressive treatment" may in particular be a treatment implementing glucocorticoids, cytostatics, interferons, antiproliferative and antimetabolic drugs (rapamycin, everolimus, mycophenolate mofetil, mycophenolic acid), inhibitors of calcineurin, in particular for their anti-graft rejection action (ciclosporin, tacrolimus (FK506), voclosporin), S1P-R agonists (FTY720), malononitrilamides (FK778), antibodies such as, for example, antithymocyte globulin or certain monoclonal antibodies directed against specific antigens such as muromonab-CD3, daclizumab, basiliximab, rituximab, alemtuzumab, infliximab, adalimumab, efalizumab.

The term “immunosuppressive treatment” also means radiotherapy or chemotherapy.

According to a preferred embodiment, the description relates to an in vitro or ex vivo method for evaluating a risk of complication in a patient not taking immunosuppressive treatment admitted to a hospital, in particular to the emergency department, intensive care unit, intensive care unit or continuing care unit.

There are several departments within a hospital depending on the patient's medical situation. By "emergency service" is meant the service which concerns the reception of the sick and injured who present themselves spontaneously or brought by ambulances or firefighters' rapid-rescue vehicles. The role of the emergency department is to welcome without selection twenty-four hours a day, every day of the year, any person presenting themselves in an emergency situation, including psychiatric, and to take care of them, in particular in case of vital distress and emergency.

By “resuscitation service” we mean the specialized service where the most serious patients are hospitalized. They benefit from constant monitoring of vital functions such as ventilation, oxygenation, blood pressure, cardiac and renal functions. If necessary, an assistance of these vital functions can be put in place in order to allow the patient's survival if possible. Patients are admitted to intensive care if they have a failure of a vital function such as, for example, during a serious infection (septic shock), drug intoxication, polytrauma, coma, acute renal failure, acute respiratory failure, after cardiac arrest or postoperatively after major surgery such as cardiac or digestive surgery.

By "intensive care unit" or "intensive care service" or "ICU" is meant the service whose mission is to take care of patients in critical condition, that is to say who present a failure of at least least a vital function, or who are at risk of developing a severe complication. The intensive care unit has very specialized technical means. These are implemented on an ongoing basis by a multidisciplinary team in order to detect, prevent and correct acute and presumed reversible imbalances related to the underlying condition (illness, surgery, trauma, intoxication, burns, sepsis).

By "continuing care unit" or "continuous monitoring unit" or "USC", we mean a hospital service intended to receive and take care of patients requiring close monitoring. The continuous monitoring units take care of patients whose condition and treatment raise fears of the occurrence of one or more vital failures patients whose condition, on emerging from one or more vital failures (after a stay in intensive care , for example), is too severe or unstable to allow return to a conventional inpatient unit. Continuous monitoring units constitute an intermediate level between resuscitation services on the one hand and conventional care services on the other. According to a preferred embodiment, the present in vitro or ex vivo evaluation method makes it possible to evaluate a risk of complication in a patient admitted to an intensive care unit.

The present evaluation method is more particularly suited to the evaluation of a risk of complication in a patient who has suffered an immuno-inflammatory attack.

By “immuno-inflammatory attack” we mean a trauma or injury that has the effect of inducing a hyper-inflammatory response in the patient’s body. The persistence of the latter leads to acquired immunosuppression, linked to an anti-inflammatory profile, and ultimately the inability to control infections. This phenomenon, also known as immunoparalysis, is characterized by the downregulation of the expression of major histocompatibility complex class II molecules on the surface of monocytes, but not B cells. monocyte/macrophage function reduces the release of immune complexes, impairs antigen-presenting abilities, and decreases natural killer cell (NK) cell function. This type of reaction is common in non-immunocompromised patients in intensive care units (Hotchkiss et al., 2013).

An "immuno-inflammatory attack" includes in particular trauma for patients with trauma, burns for patients with burns, surgery for patients who have undergone surgery or sepsis for patients in a septic state.

According to a preferred embodiment, the description relates to an in vitro or ex vivo method for evaluating a risk of complication in a patient not taking immunosuppressive treatment, said patient being in a septic state, more particularly in septic shock, and/or a patient with burn(s), more particularly severe burn(s), and/or a patient with trauma(s), more particularly severe trauma(s), and/or a patient with undergone a surgical operation, more particularly a major surgical operation.

By “sepsis patient” or “sepsis patient” is meant a patient with at least one life-threatening organ failure caused by an inappropriate host response to an infection.

By "septic shock" is meant a subtype of sepsis, in which hypotension persists, despite adequate vascular filling. “Patient with burn(s)” means that the patient has cellular destruction of the skin and underlying structures caused by a thermal burn and/or an electrical burn and/or a chemical burn and/or a burn by radiation. A burn can be superficial, intermediate, or deeper and have a generalized or particular location such as the neck, face, eyes, hands, feet, joints or other parts of the body.

By "severe burn(s)" or "patient suffering from severe burn(s)" is meant a patient whose burnt body surface is greater than 15% of the total body surface, preferably greater than 20%, preferably greater than 25%, and very particularly greater than 30% of the total surface of the body.

By “patient with trauma(s)” is meant a patient admitted directly to an intensive care unit. “Patient with severe trauma(s)” means a patient with an injury severity score (ISS, injury severity score, Baker et al, 1974) greater than 15, preferably greater than 20 and more preferably, greater than 25.

"Surgical patient" means a patient who has undergone an invasive surgical procedure intended to treat a medical condition such as disease or injury, to aid or improve bodily function, appearance, or to repair injured areas.

"Major surgery" or "patient who has undergone major surgery" means a surgical procedure presenting a technical difficulty and/or presenting a haemorrhagic risk and/or presenting a risk of death and/or long duration (such as example greater than 3 hours, preferably greater than 4 hours such as for example 5 hours or 6 hours, or even more), and/or requiring significant post-operative care. By way of non-limiting example, major surgery means surgery planned for at least one of the following indications: esophago gastrectomy, Bricker's bladder resection (total resection of the bladder with reconstruction from the small intestine) , cephalic pancreaticoduodenectomy (Whipple procedure) and/or abdominal aortic aneurysm surgery by laparotomy.

Some preferred embodiments are described below. This description is not limited to these embodiments and other particular embodiments may be implemented by combining one or more of the characteristics described previously.

The inventors therefore studied the phenomena of viral reactivation in patients in healthcare establishments. More specifically, they measured the presence and/or the variation of the viral load in TTV in biological samples from said patients at different times during their care in a healthcare establishment.

The inventors have in particular observed that the viral load could change over time depending on the patient. In particular, they surprisingly found that this change is an indicator of a risk of occurrence of a healthcare-associated infection and/or a risk of extending the duration of treatment. Thus, the inventors have developed a process for in vitro or ex vivo evaluation of the risk of complications in a patient admitted to a healthcare establishment.

The method described here is based on measuring the viral load in TTV. By “torque teno virus” or “Torque Teno Virus” or “TTV” is meant here a virus of the Anelloviridae family. A TTV as understood here is a non-enveloped virus, with a circular single-stranded DNA genome of negative polarity.

By "TTV genome", it is here referred to all the genomes of all the Anelloviridae, and in particular the genomes of the genera Alphatorquevirus (TTV), Betatorquevirus (TTMV), and Gammatorquevirus (TTMDV) found in humans. By way of example, reference is made here to the genome of the prototype strain of the Torque Teno virus, TTV-1a. More specifically, an example of a TTV genome is a sequence such as for example that which is represented by SEQ ID No 1 and whose Genbank accession number is AB017610.

In 1997, by gene subtraction method, a viral sequence fragment of about 500 base pairs (bp) was discovered in the serum of a Japanese patient with post-transfusion hepatitis of undetermined etiology (1-7) . This clone, initially named N22 and not listed in viral sequence databases at the time, was renamed TT virus (TTV), after the initials of the patient in whom it was discovered (Nishizawa et al., 1997). Further work subsequently showed that the TTV genome consists of single-stranded circular DNA of negative polarity. It is the first single-stranded circular DNA virus isolated in humans. The use of primers making it possible to amplify sequences in this non-coding region showed a high prevalence of TTV in the world population (around 90%).

TTV produces chronic infections with no clearly associated clinical manifestation. We speak of an apathogenic or orphan virus. Numerous studies have thus focused on the involvement of TTV in human pathology, in particular in certain hepatic pathologies, without a clear role being identified for this virus.

Preferably, the TTV genome has a size of about 3.8 kb. The structure and genomic organization of TTVs is well known to those skilled in the art (cf. for example refer to Biagini, Curr Top Microbiol Immunol.ll'l: 21-33, 2009) and is exemplified in [Fig 1 ].

Thus, the TTV genome can be divided into an untranslated region (UTR) of approximately 1-1.2 kb and a potential coding region of approximately 2.6-2.8 kb. The coding region contains in particular two large open reading frames: ORF1 and ORF2, coding two proteins of 770 and 202 residues respectively. In the TTV genome represented by SEQ ID No 1, the open reading frames ORF1 and ORF2 are between nucleotides 589-2901 and 107-715, respectively. The TTV genome may have other open reading frames. For example, the TTV genome may include two additional reading frames, ORF3 and ORF4 [Fig 1].

The UTR untranslated region is well conserved. It comprises in particular a GC-rich sequence capable of forming a secondary structure. The amplification of selected sequences in the UTR-5' untranslated region has demonstrated that the prevalence of the virus is very high throughout the entire world population (Hu et al., J Clin Microbiol. 43(8) : 3747-3754, 2005). This region comprises in particular a sequence of 128 bp and can be amplified by methods known to those skilled in the art, such as for example the TTV R-GENE® diagnostic kit (bioMérieux, France).

By “viral load”, we mean the number of viral particles present in a biological sample. Viral load reflects the severity of a viral infection. The viral load can be determined by measuring the amount of one of the components of the virus (genomic DNA, mRNA, protein, etc.) in the biological sample.

Preferably, the viral load thus refers to the proportion of nucleic acid sequences belonging to said virus in a biological sample. More preferentially, the viral load represents the number of copies of the genome of said virus in a biological sample.

In the present case, the viral load represents the TTV load. The “TTV load” corresponds here more particularly to the TTV viral load, that is to say the number of TTV viral particles present in a biological sample. TTV load in a patient means the viral load of any TTV harbored by said patient. The TTV load can be determined by measuring the amount of a TTV component in this biological sample.

Preferably, the TTV load corresponds to the quantity of TTV nucleic acid sequences present in a biological sample. Thus, the determination of the TTV load in a patient according to the method of the invention comprises the estimation of the number of sequences of all the TTVs in a biological sample of said patient. In particular, there is no selection, according to the method of the invention, of specific strains of TTV to be measured in said biological sample. Indeed, the detection of a high viral load in TTV and particularly, the detection of a variation in the load in TTV, is associated with an increased risk of complication, in particular of occurrence of an infection associated with care, independently of the strain or strains of Anelloviridae detected.

Preferably, the determination of the TTV load comprises the determination of the quantity of active and/or inactive viral copies. It consists of determining the quantity of circulating, integrated or latent viral copies.

In particular, it is possible to determine the number of viral copies of TTV, by previously determining the detection limit of TTV.

By “limit of detection” or “LOD”, is meant the smallest quantity of copy of the genome that can be distinguished from an absence of detection (blank value) using viral standards. Then, by extrapolation from standard curves, it is then possible to determine a viral load in copies of viral DNA per pL in the reaction tube, then in copies of viral DNA per mL in said sample.

In the context of the present description, a high TTV viral load is considered to be a viral load in the biological sample greater than 7,000 copies per mL of biological sample such as, for example, blood or one of its derivatives. such as plasma and/or serum, preferably greater than 10,000 copies per mL of plasma, preferably greater than 20,000 copies per mL of sample biological, and more preferably, greater than 40,000 copies per mL of biological sample.

TTV levels - and therefore TTV load - can be determined according to methods well known to those skilled in the art by measuring levels of TTV DNA, TTV RNA or TTV proteins. The method according to the invention can thus comprise between the taking of the sample from the patient and step a) as defined below, another preliminary step corresponding to the transformation of the biological sample into a DNA sample. double-stranded, or into a sample of mRNA (or corresponding cDNA), or into a sample of proteins, which is then ready to be used for the in vitro measurement of the viral load in TTV in step a) as as defined below.

The preparation or extraction of viral double-stranded DNA, mRNA (as well as the reverse transcription of this into cDNA) or proteins from a cell sample are only well-known routine procedures of the person in the trade.

The double-stranded DNA can correspond either to the entire TTV genome, or only to a part of it. Once a ready-to-use dsDNA, mRNA (or corresponding cDNA) or protein sample is available, detection of TTVs can be performed, depending on the type of sample or transformation, either by genomic DNA (i.e. based on the presence of at least one sequence consisting of at least part of the TTV genome), or by mRNA (i.e. based on the TTV mRNA content in the sample), or at the protein level (i.e. based on the TTV protein content in the sample).

Preferably, TTV levels are determined by measuring levels of TTV nucleic acid, more preferably TTV DNA.

Methods for detecting a nucleic acid in a biological sample are well known to those skilled in the art and include, among others, amplification, preferably PCR amplification, more preferably real-time PCR, sequencing, hybridization with a labeled probe.

Advantageously, the description therefore relates to a method for in vitro or ex vivo evaluation of the risk of complications in a patient not taking immunosuppressive treatment admitted to a health establishment, comprising measuring the viral load of at least one torque teno virus (TTV) in a biological sample from said patient, said viral load being measured by amplification, sequencing or hybridization of at least one TTV sequence preferably by amplification, more preferably by real-time PCR.

According to a first embodiment, the TTV load is determined by amplifying the TTV sequences.

According to this embodiment, a preferred approach consists in amplifying sequences which are known to be specific for the TTV genome, and in particular preferably at least one sequence of the UTR untranslated region of the TTVs as defined previously.

By “specific sequence of the TTV genome”, is meant here a sequence which is present in the majority of the known TTVs, but which is absent from the majority of the other viruses, in particular from the majority of the other anelloviruses. Preferably, a TTV specific sequence is present in at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of known TTV genomes. More preferably, it is present in 100% of known TTV genomes. Alternatively, a TTV-specific sequence is present in less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of known non-TTV anellovirus genomes. Preferably, a TTV-specific sequence is absent from all known non-TTV anellovirus genomes. Particularly preferably, such a sequence is for example a sequence comprised in the UTR untranslated region which is well conserved from one TTV to another. More particularly, such a sequence can correspond to the 128 bp sequence of the 5'-UTR untranslated region which can be amplified according to methods known to those skilled in the art, for example using the TTV R diagnostic kit - GENE® (bioMérieux, France).

Thus according to this embodiment, the method described here comprises the use of primers and probes for the amplification of sequences known to be specific for the TTV genome. As usual in this technical field, these primers are preferably oligonucleotides. For example, these primers can comprise less than 30 nucleotides, at least 25 nucleotides, less than 20 nucleotides, less than 15 nucleotides or less than 12 nucleotides. Alternatively, these primers comprise at least 12, 15, 20.25 or 30 nucleotides. Preferably, the primers used comprise between 12 and 20 nucleotides, between 12 and 25 nucleotides, between 15 and 20 nucleotides or even between 15 and 25 nucleotides. The A person skilled in the art knows how to determine the length and the sequence of the primers to be used to amplify the specific sequence(s) of the TTVs. It could, for example, use the same primers as those supplied in the TTV R-GENE® diagnostic kit (ref. 423414 and 69-030B, bioMérieux, France).

The amplification techniques include in particular isothermal methods and techniques based on PCR (Polymerase Chain Reaction). Isothermal amplification methods include a large number of methods. The most used to detect pathogens are the LAMP (Loop-Mediated Amplification) and RPA (Recombinase Polymerase Amplification) methods. Isothermal amplification methods also include methods such as, for example, NASBA (nucleic acid sequence-based amplification), HDA (helicase-dependent amplification), RCA (rolling circle amplification) and SDA (strand displacement amplification), EXPAR ( exponential amplification reaction), ICANs (isothermal and chimeric primer-initiated amplification of nucleic acids), SMART (signal-mediated amplification of RNA technology), etc. (see for example Asiallo and Baeumner, Lab Chip 11(8): 1420-1430, 2011).

Preferably, the PCR technique used quantitatively measures the initial amounts of DNA, cDNA or RNA. Examples of PCR-based techniques that may be used in the methods described herein include, without limitation, techniques such as real-time PCR (Q-PCR), reverse PCR (RT-PCR), multiplex reverse PCR , real-time reverse PCR (QRT-PCR) and digital PCR (or digital PCR). These techniques are well known technologies and readily available to those skilled in the art.

Preferably, the determination of the TTV load is carried out by real-time quantitative PCR. Numerous methods for the detection and quantification of TTVs have been described in the art (see for example Maggi et al., J Virol. 77(4): 2418-2425, 2003).

Reference will be made in particular to the method described by Kulifaj et al. (J Clin Virol. 105:118-127, 2018). This method is particularly advantageous due to its simplicity and robustness. It is based on the amplification of a sequence included in the non-coding region UTR. This sequence is present in all known TTVs, thus conferring a very high specificity to the method. In addition, it is particularly versatile and can be implemented with any type PCR platform. It is particularly advantageous to use the TTV R-GENE® diagnostic kit (ref. 423414 and 69-030B bioMérieux, France) to implement this method.

Alternatively, viral load determination is performed by digital PCR. Digital PCR involves multiple PCR runs on extremely dilute nucleic acids such that most positive amplifications reflect the signal from a single template molecule. Digital PCR thus allows the counting of individual model molecules. The proportion of positive amplifications among the total number of PCRs analyzed allows an estimate of the template concentration in the original or undiluted sample. This technique has been proposed to allow the detection of a variety of genetic phenomena (Vogelstein et al., Proc Natl Acad Sci USA 96: 9236-924, 1999). Digital PCR, like real-time PCR, potentially allows the discrimination of fine quantitative differences in target sequences between samples.

In another embodiment, TTV DNA levels are measured by sequencing. As used herein, the term "sequencing" is taken in its broadest sense and refers to any technique known to those skilled in the art for determining the sequence of a polynucleotide molecule (DNA or RNA), i.e. that is to say, to determine the succession of nucleotides composing this molecule.

TTV DNA can thus be sequenced by any technique known in the art. Sequencing as understood herein includes but is not limited to Sanger sequencing, whole genome sequencing, hybridization sequencing, pyrosequencing (including 454 sequencing, Solexa Genome Analyzer sequencing), sequencing with capillary electrophoresis, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, massively parallel signature sequencing (MPSS), reversible dye terminator sequencing, matched pairs, short-term sequencing, sequencing with exonucleases, sequencing by ligation, single molecule sequencing, sequencing by synthesis, sequencing by electron microscopy real-time sequencing, reverse-termination sequencing, sequencing by nanopores, reversible terminator sequencing, semiconductor sequencing, SOLiD(R) sequencing, Single Molecule Real-Tim (SMRT) sequencing e Analysis), MS-PET sequencing, mass spectrometry, and their combinations. A particular embodiment uses sequencing high throughput of DNA, using for example the MiSeq, NextSeq 500 platforms, and the HiSeq series developed by Illumina (Reuter et al., Mol Cell, 58: 586-597, 2015; Bentley et al. Nature; 456 : 53-59, 2008), the Genome Sequencer platform from 454 and Roche (Margulies et al. Nature; 437: 376-380, 2005), the SOLiD platform from Applied Biosystems (McKernan et al., Genome Res; 19: 1527-1541, 2009), the Polanator platform (Shendure et al., Science, 309: 1728-1732) or even the Helicos single molecule sequencing platform (Harris et al. Science; 320: 106-109, 2008). High-throughput sequencing also includes methods such as SMRT real-time sequencing (Roads et al., Genomics, Proteomics & Bioinformatics, 13(5): 278-289, 2015), Ion Torrent sequencing (WO 2010/008480; Rothberg et al., Nature, 475: 348-352, 2011) and sequencing using nanopores (Clarke J et al. Nat Nanotechnol: 4: 265-270, 2009).

Sequencing is performed on all of the DNA contained in the biological sample or on parts of the DNA contained in the biological sample. It is immediately clear to the person skilled in the art that said sample contains at least a mixture of TTV DNA and host subject DNA. Additionally, TTV DNA will likely represent only a minor fraction of the total DNA present in the sample. Advantageously, the DNA is randomly fragmented, generally by physical methods, prior to sequencing.

A first approach consists in sequencing specific sequences of the genome of a TTV species. Another approach is to use a method that allows quantitative genotyping of nucleic acids obtained from the biological sample with high precision. In a particular embodiment, precision is achieved by analyzing large numbers (e.g., millions or billions) of nucleic acid molecules without any amplification using protocols that rely on knowledge beforehand of the target sequences (that is to say in this case, the sequences of the TTVs).

In a preferred embodiment, the method of the invention comprises a step of quantifying the number of readings.

In a particular embodiment, a random subset of nucleic acid molecules from the biological sample is subjected to high-throughput sequencing. Preferably, the TTV sequences are identified in the global sequencing data by comparison with publicly deposited TTV sequences. That comparison is advantageously based on the level of sequence identity with a known TTV sequence and makes it possible to detect even remote variants. Common software such as BLAST can be used to determine the level of identity between sequences.

Thus, a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with a known TTV sequence is identified as a TTV sequence. According to this embodiment, the determination of the TTV load therefore comprises the numbering of the TTV sequences identified by sequencing in the biological sample of the subject.

In another embodiment, the TTV load is determined by measuring the amount of a viral protein in the biological sample. It is thus possible to use specific antibodies, in particular in well-known technologies such as immunoprecipitation, immunohistology, western blot, dot blot, ELISA or ELISPOT, electrochemiluminescence (ECLIA) , protein arrays, antibody arrays, or tissue arrays coupled with immunohistochemistry. Among the other techniques which can be used are included FRET or BRET techniques, microscopy or histochemistry methods, including in particular confocal microscopy and electron microscopy methods, methods based on the use of one or several excitation wavelengths and a suitable optical method, such as an electrochemical method (the techniques of voltammetry and amperometry), the atomic force microscope, and radiofrequency methods, such as multipolar, confocal resonance spectroscopy and non-confocal, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or index of refraction (for example, by surface plasmon resonance, or "surface plasmon resonance" in English, by ellipsometry, by method resonant mirror, etc.), flow cytometry, radioisotopic or magnetic resonance imaging, analysis by polya gel electrophoresis crylamide (SDS-PAGE); by mass spectrometry and by liquid chromatography coupled to mass spectrometry (LC-MS/MS). All these techniques are well known to those skilled in the art and it is not necessary to detail them here. Furthermore, antibodies specific for TTV proteins are already available. In a preferred embodiment, the method described here includes an additional step of normalizing the amount of nucleic acid or viral protein measured.

According to a preferred embodiment, it may be advantageous to normalize the levels of TTV, i.e. the amount of TTV DNA, TTV RNA or TTV proteins present in the biological sample to a specific parameter of this sample.

Normalizing the measured TTV load to a specific parameter reduces the error rate when comparing viral loads from two different biological samples. An example of a parameter that can be useful for this normalization can be a physical parameter, independent of the content of the sample, such as its volume, for example. It is also possible to normalize the amount of TTV DNA, TTV RNA or TTV protein to the total amount of DNA, RNA or protein present in the sample. Alternatively, it is possible to use a particular DNA or RNA sequence or a particular protein as a standardization tool. For example, this sequence or this protein can be a human sequence or protein.

Alternatively, the amount of TTV DNA or RNA or TTV protein in a given sample is compared to an internal control.

For this, the quantity of nucleic acid or of TTV protein measured in the biological sample can be related to a defined quantity of a nucleic acid or of an appropriate protein which can be identified and quantified, such as an acid nucleic acid or a host or exogenous protein. Preferably, this identifiable and quantifiable nucleic acid or protein is processed (eg, amplified, sequenced, etc.) as the target nucleic acid or protein.

It is thus possible to add ab initio to the sample a known quantity of this nucleic acid or this identifiable and quantifiable protein, which will then be treated as the nucleic acid or the target protein and will go through all the stages of preparation of the sample. before measuring the amount of this nucleic acid or this viral protein. The preparation steps can include means to protect the viral nucleic acid and destroy the host nucleic acids, for example by using different nucleases.

Alternatively, these steps may include means to protect viral proteins and destroy host proteins, for example by using different proteases. The internal control makes it possible to evaluate the quality and the extent of the processing (for example an amplification or a sequencing) of the molecules considered (nucleic acids or proteins) in the sample. Preferably, said internal control is a nucleic acid molecule of known sequence, this nucleic acid molecule being present in the sample at a known concentration. More preferably, this nucleic acid molecule is the genomic single-stranded circular DNA molecule of a virus of known sequence and concentration in the sample. Such a known virus may for example be a virus from the Circoviridae family. The ratio of the number of sequences of the sample to the control makes it possible to estimate the absolute number of TTV genomes of known sequence and concentration. Alternatively, this internal control is a protein of known sequence, which is present in the sample at a known concentration.

Once the TTV load has been determined by measuring the quantity of nucleic acid or TTV protein determined, this being optionally normalized, it may be advantageous to compare it with a predetermined reference TTV load.

According to this preferred embodiment, the description relates to an in vitro or ex vivo method for evaluating a risk of complication in a patient not taking immunosuppressive treatment admitted to a healthcare establishment, comprising the implementation of the steps consisting of: a) measuring the viral load of at least one TTV in a biological sample from said patient, b) comparing the viral load determined for said biological sample with a predetermined reference value, and c) establishing a risk of complication when the viral load determined for said sample is higher or lower than the predetermined reference value.

By “a reference TTV load” or “a reference viral load” is meant within the meaning of the present application any TTV load used as a reference.

This means that the reference TTV load corresponds to “a reference level of nucleic acid (or protein) of TTV” or “a control level of nucleic acid

(or protein) of TTV”, that is to say at a concentration of a nucleic acid (or protein) of TTV used as a reference. As used herein, "a TTV nucleic acid (or protein) reference concentration" is a baseline level measured in a control sample comparable to that tested, and which is obtained from a subject or group of subjects with a particular traumatic and/or infectious state. It may be for example a subject or a group of subjects who are healthy or do not exhibit any particular trauma and/or infection. It may also be a patient or a group of patients admitted to a healthcare establishment, in particular an intensive care unit, in particular a patient or a group of patients who have suffered an immuno-inflammatory attack. such as a patient or a group of patients suffering from sepsis, suffering from burn(s), trauma(s) and/or having undergone surgery. Finally, it may be the same individual having undergone surgery, for example before or just after it. The reference level can be determined by a plurality of methods. For example, the control can be a predetermined value, which can take various forms. It can be a single threshold value, such as a median or mean. The “reference level” can be a single value, also applicable to each patient individually. Alternatively, the baseline may vary based on specific patient subpopulations. So, for example, older men might have a different baseline than younger men for TTV load, and women might have a different baseline than men for this viral load. On the other hand, the "reference level" can be established on the basis of comparative groups, such as groups not having a high level of TTV nucleic acid (or protein) and groups having levels of nucleic acid (or protein) of elevated TTVs. Another example of comparison groups would be groups with a particular disease, condition, or symptoms and groups without disease. The predetermined value may be set, for example, when a test population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group, and a high-risk group.

The reference level can also be determined by comparing the level of nucleic acid (or protein) of TTV in populations of patients suffering from diseases or taking a treatment leading to a state of immunosuppression. This can be accomplished, for example, by histogram analysis, in which an entire cohort of subjects is presented graphically, with a first axis representing the level of said TTV nucleic acid (or protein), and a second axis represents the number of patients in the patient group expressing TTV nucleic acid (or protein) at a given level. Two or more distinct groups of subjects can be determined by identifying subpopulations of the cohort that have the same or similar TTV nucleic acid (or protein) levels. The determination of the reference level can then be made on the basis of a level that best distinguishes these distinct groups. A reference level can also represent the levels of two or more of the present TTV nucleic acids (or proteins). Two or more markers can be represented, for example, by a ratio of values for the levels of each marker.

Similarly, an apparently healthy population will have a different 'normal' range than a population known to exhibit a condition associated with a high concentration of said TTV nucleic acid (or protein). Accordingly, the selected predetermined value may take into account the category into which an individual falls. Appropriate ranges and categories can be selected with just routine experimentation by the skilled person. By raised , augmented , we mean raised relative to a selected control. Generally, control will be based on normal, apparently healthy individuals in an appropriate age range.

In a preferred embodiment, the reference concentration corresponds to the concentration of TTV nucleic acid (or protein) or combination of TTV nucleic acids (or proteins) in the general population.

It will also be understood that the controls in the method described here can be, in addition to predetermined values, biological samples measured in parallel with the samples tested. According to this embodiment, the reference level will be that of the TTV nucleic acid(s) (or proteins) in a sample from a healthy subject.

Preferably, the reference concentration of TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or this protein) in a healthy subject or in a population of healthy subjects.

According to another preferred embodiment, the reference concentration of the nucleic acid (or protein) of TTV is the concentration of this nucleic acid (or this protein) of TTV in a patient who has suffered an immuno-inflammatory attack or in a group of patients who suffered an immuno-inflammatory attack. According to another preferred embodiment, the reference concentration of the nucleic acid (or protein) of TTV will be the concentration of this nucleic acid (or this protein) of TTV in a patient suffering from sepsis or in a group of patients with sepsis.

According to another preferred embodiment, the reference concentration of the TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or this protein) in a patient suffering from burn(s) or in a group of patients suffering from burn(s).

According to another preferred embodiment, the reference concentration of the nucleic acid (or protein) of TTV is the concentration of this nucleic acid (or this protein) of TTV in a patient suffering from trauma(s) or in a group of patients suffering from trauma.

According to another preferred embodiment, the reference concentration of the nucleic acid (or protein) of TTV the concentration of this nucleic acid (or this protein) of TTV in a patient having undergone surgery or in a group of patients having undergone surgery.

According to another preferred embodiment, the reference concentration of the nucleic acid (or protein) of TTV the concentration of this nucleic acid (or this protein) of TTV in the same individual having undergone the surgery at a specific time, for example before or just after this one.

By comparing the viral load of TTV in the biological sample of said patient with a predetermined reference value, it is then possible to establish the risk of complications in said patient.

According to a particular embodiment, the predetermined reference value corresponds to a viral load in the patient's biological sample, for example blood or one of its derivatives such as plasma and/or serum, greater than 7,000 copies per ml_ of biological sample, for example greater than 10,000 copies, 20,000 copies or even 40,000 copies per ml_ of biological sample. According to this particular embodiment, a higher TTV viral load in the patient than the predetermined reference value is correlated with the severity of the patient's condition and may also be correlated with an increased risk of complication, in particular the occurrence of healthcare-associated infection. According to a variant of this particular embodiment, the predetermined reference value corresponds to a viral load in TTV of 10,000 copies per ml of biological sample. Thus, according to this variant, it is established that there is an increased risk of complication when the TTV viral load determined in the patient's biological sample is greater than said predetermined reference value. Conversely, it is established that there is no increased risk of complication when the TTV viral load determined in the patient's biological sample is lower than said predetermined reference value.

A higher TTV viral load in the patient compared to that of a healthy individual, or group of individuals, may be an indicator that there is an increased risk of compilation in said patient.

The term "greater", as used herein in certain embodiments, means a greater amount, e.g., an amount slightly greater than the original or reference amount, or e.g., an amount in great excess relative to the original or reference quantity, including all quantities in between. Alternatively, "increase" may refer to an amount or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more than the quantity or activity for which the increased quantity or activity is compared.

The term "lower", as used herein in certain embodiments, means a lesser amount, for example, an amount slightly less than the original or reference amount, or for example, a greatly deficient amount. relative to the original or reference quantity, including all quantities in between. Alternatively, "decrease" may refer to an amount or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% less than the quantity or activity for which the reduced quantity or activity is being compared.

Advantageously, it is also possible to use as reference TTV viral load, the viral load of at least one TTV in the same patient at a second instant. According to a preferred embodiment, the description relates to an in vitro or ex vivo method for evaluating a risk of complication in a patient not taking immunosuppressive treatment, admitted to a health establishment, comprising the implementation of steps consisting of: a) measuring a first viral load of at least one TTV in said biological sample of the patient taken from a first sample taken at time T1, b) measuring a second viral load of at least one TTV in said sample of the patient from a second sample taken at time T2, c) calculate the variation between the viral load at T2 and the viral load at T1, giving an ATTV value, d) establish a conclusion as to the risk of complication, from of the results of the comparisons.

Surprisingly, the inventors have determined that a variation in the viral load in TTV in the patient during his stay in a health establishment, namely an increase or a decrease in the viral load of TTV according to the values defined above -after, is correlated with an increased risk of complications in said patient, in particular an increased risk of developing a healthcare-associated infection.

This method is thus particularly useful for evaluating and monitoring over time the risk of complications in a patient admitted to a healthcare establishment. In a preferred embodiment, the first sample of step a) is taken at a time T1 chosen from: the day of admission, the ^1st day, the ^2nd day, the ^3rd day, the ^4th day, the ^5th day, the ^6th day and the ^7th day, it being understood that the first day corresponds to the day after the day of admission and/or the day after the day on which the said patient suffered an immuno-inflammatory attack.

In a particular embodiment, when said patient is a patient admitted to a health establishment for surgery, more particularly for major surgery, the first sample of step a) can be taken at the time of the surgery , that is to say just before, just after or during the surgical act.

In a preferred embodiment, the second sampling of step b) is carried out at a time T2, this second time being later than the first time T1 of step a). Preferably, the second sample is taken between the ^8th day and the ^31st day, and preferably still, between the ^14th day and the ^31st day. Thus, the time T2 is preferably chosen from: the ^14th day, the ^15th day, the ^16th day, the ^17th day, the ^18th day, the ^19th day, the ^20th day, the ^21st day , ^22nd day, ^23rd day, ^24th day, ^25th day, ^26th day, ^27th day, ^28th day, ^29th day, ^30th day and ^31st day .

In another embodiment, the second sample is taken after the ^31st day, such as at least 40 days, 60 days, 90 days, 100 days, 120 days, 150 days, 180 days, 210 days, 240 days , 270 days, 300 days, 330 days, 360 days after admission of said patient to a health facility.

In certain embodiments, the measurement of the viral load in TTV is also carried out on a plurality of samples taken at different times comprised between times T1 and T2. In particular, the measurement of the viral load in TTV is also carried out on at least 1 sample, preferably at least 2 samples, preferably at least 3 samples, preferably at least 4 samples, preferably at least 5 samples, such as for example 6, 7, or 8 samples taken at different times between times T1 and T2.

According to certain embodiments, the viral load in TTV at time T1 corresponds to the mean or the median of the viral loads measured in a plurality of biological samples from said patient, taken between the day of admission, and/or immuno-inflammatory attack, and on the ^7th day. Preferably, the viral load in TTV at time T1 corresponds to the average or the median of the viral loads measured in at least two, preferably at least three, preferably at least four biological samples from said patient, taken between the day of admission, and/or immuno-inflammatory attack, and on the ^7th day. By way of non-limiting example, the viral load in TTV at time T1 may correspond to the mean or the median of the viral loads measured in biological samples from said patient taken on days 1 or 2, and on days 3 or 4, and on 5, 6 or 7 days.

According to certain embodiments, the viral load in TTV at time T2 corresponds to the mean or the median of the viral loads measured in a plurality of biological samples from said patient, taken between the ^8th day following admission, and/or from immuno-inflammatory attack, and the ^31st day. Preferably, the viral load in TTV at time T2 corresponds to the average or the median of the viral loads measured in at least two, preferably at least three, preferably at least four biological samples from said patient, taken between the ^8th day following admission, and/or the immuno-inflammatory attack, and the ^31st day. By way of non-limiting example, the viral load in TTV at time T2 may correspond to the average or the median of the viral loads measured in biological samples of said patient taken on the ^14th day, or any day between the ^13th and on the ^18th day, and on the ^28th day, or any day between the 26th and ^36th ^day .

Advantageously, it is then possible to calculate in step c) the ATTV value corresponding to the variation between the viral load in TTV at time T2 and the viral load at time T1, i.e.:

ATTV = (viral load in TTV at time T2) - (viral load in TTV at time T1)

Depending on the viral load in TTV at time T1 and the viral load in TTV at time T2, the person skilled in the art is able to calculate the slope of the straight line defined by these two measurements. The ATTV thus corresponds to the value of the slope between times T1 and T2.

According to the present description, it will preferably be considered that the viral load in TTV between times T1 and T2 is stable if the ATTV value is included in the range going from -1.25 to +1.25, preferably in the range going from -1.20 to +1.20, preferably in the range from -1.15 to +1.15, preferably in the range from -1.10 to +1.10. For example, viral load stability may correspond to a slope in the range of -1.25 to +1.25 measured between median viral loads for a plurality of samples at T1 and T2.

In addition, it will preferably be considered that the viral load in TTV between times T1 and T2 is increased if the ATTV value is greater than +1.25, preferably greater than +1.35, preferably greater than +1.40 , preferably greater than +1.45, and more preferably greater than +1.50.

In addition, it will preferably be considered that the viral load in TTV between times T1 and T2 is reduced if the ATTV value is less than -1.25, preferably less than -1.35, preferably less than -1.40 , preferably less than -1.45, and more preferably less than -1.50.

Therefore, if the viral load change in TTV between time T1 and T2 (ATTV) is in the range from -1.25 to +1.25, it can be concluded in step d) that the patient is not at increased risk of a healthcare-associated infection.

Conversely, if the variation in viral load in TTV between times T1 and T2 (ATTV) is not included in the range going from -1.25 to +1.25, it can be concluded in step d) the patient is at increased risk of developing a healthcare-associated infection.

Thus, in a preferred embodiment, the method according to the present description comprises the implementation of the steps consisting in: a) measuring a first viral load of at least one TTV in said biological sample of the patient taken from a first sample taken at time T1, b) measuring a second viral load of at least one TTV in said biological sample of the patient taken from a second sample taken at time T2, c) calculating the variation between the viral load at T2 and the viral load at T1 , giving an ATTV value, d) draw a conclusion as to the absence of an increased risk of complication when the ATTV value is in the range from -1.25 to +1.25, preferably - 1.20 to +1.20, and more preferably from −1.10 to +1.10.

According to a particularly advantageous embodiment, the description relates to an in vitro or ex vivo method for evaluating a risk of complication in a patient not taking immunosuppressive treatment, admitted to a health establishment, comprising measuring the viral load of at least one torque teno virus (TTV) in a biological sample from said patient and further comprising detecting the presence of at least one herpesvirus (HPV) in said biological sample from the patient.

Indeed, it is to the merit of the inventors to have determined that the co-detection of a high viral load of TTV and of at least one HPV in the biological sample of the patient, in particular during the first week following his admission to intensive care, is an indicator of an increased risk of complications in said patient, in particular an increased risk of developing a healthcare-associated infection. The link between early viremia (from the first week after admission) for TTV/Hpv and the existence of an increased risk of developing HAI during the first month is also particularly surprising. The term "determination of the presence of at least one herpesvirus" or "detection of at least one herpesvirus" as used herein encompasses qualitative and/or quantitative detection. In various embodiments, detection of HPV is accomplished by detecting levels of HPV nucleic acid, more preferably HPV DNA.

According to the present description, a herpesvirus is determined as present or as detected if at least one protein of at least one strain of HPV, more preferably at least one antigen of at least one strain of HPV is detected in at least one biological sample of said patient.

Preferably, a herpesvirus is determined as present or detected if at least one nucleic acid of at least one strain of HPV, more preferably at least one DNA of at least one strain of HPV is detected in at least one biological sample of said patient. In this case, we speak of positive viremia or positive DNAemia.

By “viremia” is meant here the level of viral particles, or the viral load, in a biological sample for a given virus. The viremia is positive when the number of viral particles detected is greater than a predefined threshold (LOD) in said biological sample for a given virus. Conversely, viremia is negative when said viral particles are not detected in said biological sample.

“DNAemia” means the detection of viral DNA in a biological sample, in particular in a sample of plasma, whole blood and/or leukocytes isolated from peripheral blood and/or in a sample of buffy coat (the fraction of a sample of unclotted blood after centrifugation that contains most of the white blood cells and platelets). Several techniques are available for the detection of DNAemia, including PCR-based techniques, hybrid capture, and branched-chain DNA analysis. When DNA of the virus studied is detected in said biological sample, the DNAemia is positive. On the other hand, the DNAemia is negative when said viral DNA is not detected in said biological sample. By “viraemic event” is meant that a given virus has been detected at least once during the study, and in particular that a positive DNAemia for a given virus has been detected in at least one biological sample from the patient during the duration of the study. According to this preferred embodiment, the patient not taking immunosuppressive treatment presents an increased risk of complication when the TTV viral load in a biological sample of said patient, for example blood or one of its derivatives such as plasma and / or serum, is greater than 7,000 copies per ml_ of biological sample, preferably greater than 10,000 copies per ml_ of biological sample, and that the presence of herpesvirus is also detected. According to this preferred embodiment, the method can thus comprise the implementation of the steps consisting in: a) measuring the viral load of at least one TTV in a biological sample from said patient, b) comparing the viral load determined for said sample biological to a predetermined reference value at 10,000 copies/ml, c) determining the presence of at least one Herpesvirus, preferably by measuring DNAemia, and d) establishing the presence of an increased risk of complication when said load viral determined for said sample is greater than the predetermined reference value and when at least one Herpesvirus is present.

According to another preferred embodiment, the description relates to an in vitro or ex vivo method for evaluating a risk of complication in a patient not taking immunosuppressive treatment, admitted to a health establishment, comprising measuring the viral load of at least one torque teno virus (TTV) in a biological sample from said patient and further comprising measuring the viral load of at least one herpesvirus in said biological sample from the patient.

In the present case, the “viral load of at least one HPV” or “load of at least one HPV” corresponds to the viral load of at least one strain of HPV, that is to say the number of viral particles of at least one strain of HPV present in a biological sample.

The load of at least one HPV in a patient means the viral load of at least one strain of HPV harbored by said patient. The HPV load can be determined by measuring the amount of a component of at least one HPV strain, such as a nucleic acid or a protein, in this biological sample. Preferably, the HPV load corresponds to the quantity of nucleic acid sequences of a given strain of HPV present in a biological sample. Thus, it will be possible, according to the invention, to differentiate the different specific strains of HPV to be measured in said biological sample. Preferably, determining the HPV load includes determining the amount of active and/or inactive viral copies. It consists of determining the quantity of circulating, integrated or latent viral copies.

The methods for detecting and/or measuring the viral load of an HPV nucleic acid in a biological sample are the same as those previously described for TTV and include, among others, amplification, preferably PCR amplification , more preferably by real-time PCR, sequencing, hybridization with a labeled probe and any other methods known to those skilled in the art.

Herpesviruses (Herpesviridae or HpV) are well known to those skilled in the art. It is a family of DNA viruses affecting various animal species including birds, fish, reptiles, amphibians and mammals, including humans. All members of the HpV family share a common structure: a relatively large, single-partite, double-stranded and linear DNA, encoding 100-200 genes, enclosed in an icosahedral protein cage called the capsid, itself enveloped in a protein coat called integument, containing both viral proteins and viral mRNAs, and a lipid bilayer membrane called the envelope. All HpVs are nuclear replicating, viral DNA is transcribed into mRNA in the nucleus of the infected cell, where it can persist in a latent form indefinitely.

Nine types of HpV are known to primarily infect humans. At least five are extremely common in humans: herpes simplex viruses 1 and 2 (HSV-1 and HSV-2, or HHV-1 and HHV-2) both can cause orolabial herpes and genital herpes; varicella-zoster virus (or HHV-3) which causes chickenpox and shingles; the Epstein-Barr virus (EBV or HHV-4) implicated in several diseases, including mononucleosis and certain cancers and the human cytomegalovirus (HCMV or CMV or HHV-5).

In humans, more than 90% of adults have been infected with at least one of these viruses and a latent form of the virus persists in almost all humans who have been infected. Less common human herpesviruses are human herpesvirus 6A and 6B (HHV-6A and HHV-6B, together referred to as HHV-6), human herpesvirus 7 (HHV-7) and Kaposi's sarcoma-associated herpesvirus (KSHV, also known as HHV-8).

By “herpesvirus” or “HPV” or “HHV” (Human Herpes Virus), is meant here a virus of the Herpesviridae family. By "HPV genome", it is herein referred to the genomes of all Herpesviridae, including those of the HSV-1, HSV-2, HHV-3, EBV, CMV, HHV-6, HHV-7 and KSHV type and more particularly those of the CMV, EBV, HHV6 and HSV-1 type.

According to a preferred embodiment, said at least one herpesvirus is selected from the group consisting of: CMV, EBV, HHV6 and HSV-1. Preferably, the herpesvirus is EBV. By way of example, reference is made here to the genome of herpesvirus strains of the CMV AD169, EBV B-95, HSV1 95 and HHV6 Z29 type. By way of non-limiting example, a CMV AD169 genome may be the one whose sequence referenced under the Genbank accession number X17403.1. Similarly, an EBV B-95 genome may be one whose sequence referenced under the Genbank accession number is V01555.2. An example of an HHV6 Z29 genome may be that whose sequence is referenced under the GenBank accession number X83413.2.

Preferably, the determination of the presence of at least one HPV and/or the measurement of the HPV viral load is carried out by real-time quantitative PCR. Many HPV detection and quantification methods have been described in the art and are known to those skilled in the art (see for example Walton et al., Plos ONE 9(6): e98819, 2014). Reference will be made in particular to the method described by Mallet et al. ( Intensive Care Medicine Exp. (2019) 7:28) and more particularly in table S1 of this article reproduced here in figures 6 and 7. This method is particularly advantageous due to its simplicity and robustness, it can also be implemented implemented with any type of PCR platform. It is based on the amplification of specific genes of each of these Herpesviruses, namely ppUL83 coding for a structural protein of CM V, the BXLF1 gene coding for an EBV thymidine kinase, the US7 gene coding for a glycoprotein of HSV-1 and the U57 gene encoding an HHV6 capsid protein. It is particularly advantageous to use R-GENE® diagnostic kits (CM V R-GENE® Ref. 69-003B, EBV R-GENE® Ref.: 69-002B, HSV1 HSV2 VZV R-GENE® Ref. : 69-004B and HW6 R-GENE® Ref.: 69-100B, bioMérieux, France) to implement this method. In a preferred embodiment, the method described herein includes an additional step of normalizing the amount of HPV nucleic acid measured. In this regard, the person skilled in the art may refer to the methods and embodiments described above concerning the normalization of the levels of TTV. These methods can easily be adapted to normalize the amount of HPV nucleic acid.

Once the load of at least one HPV has been determined by measuring the quantity of HPV nucleic acid, this being optionally normalized, it may be advantageous to compare it with a reference HPV load. In this regard, the person skilled in the art may refer to the methods and embodiments described above concerning the comparison with reference levels of TTV. These methods can easily be adapted for comparison with reference levels of HPV.

Methods of treating a patient suspected of having a risk of complication

The patient's susceptibility to the risk of complications, including infections, can therefore be determined using the methods described here, allowing the development of a specific treatment to be tailored to the patient's needs.

Advantageously, the methods as described above can therefore comprise, in all their embodiments, a healthcare management step, in particular to reduce the risk of complications and in particular the risk of occurrence of an infection associated with healthcare.

A patient identified as being at increased risk of occurrence of a healthcare-associated infection may have appropriate health care management with the aim of reducing the risk of occurrence of said healthcare-associated infection and, for example, in order to reduce the risk of developing sepsis, septic shock or the risk of death.

Thus, according to another aspect, the present description also relates to a method for treating a patient suspected of having a risk of complication, characterized in that it comprises the steps consisting in: identifying the patients presenting a risk of complication by implementing any one of the embodiments as described above and, adapt the health care management of said patient identified in the previous step to reduce the risk of aggravation.

Examples of care management include an immunomodulatory treatment adapted to the patient or a prophylactic antibiotic treatment, the two treatments can be combined and/or refer to a continuing care or resuscitation service in order to reduce the risk occurrence of a treatment-associated infection, for example reducing the risk of developing sepsis, septic shock or even the risk of death in the days following the measurement of the expression of the biomarker(s). Preferably, the immunomodulatory treatment is an immunostimulatory treatment, if the individual is determined to have an immunosuppressed status, or an anti-inflammatory treatment, if the individual is determined to have an inflammatory status. .

Among the immunostimulating treatments which can be selected, mention may be made by way of examples of the group of interleukins, in particular IL-7, IL-15 or IL-3, growth factors, in particular GM-CSF, interferons , in particular IFNγ, Toll receptor agonists, antibodies, in particular antibodies directed against immune checkpoints (for example anti-PD1, anti-PDL1, anti-LAG3, anti-TIM3, anti-IL-10 or anti-CTLA4), transferrins and molecules that inhibit apoptosis, FLT3L, Thymosin a1, adrenergic antagonists.

Among the anti-inflammatory treatments, mention may be made in particular of the group of glucocorticoids, cytostatic agents, molecules acting on immunophilins and cytokines, molecules blocking the IL-1 receptor and anti-TNF treatments. Examples of appropriate prophylactic antibiotic treatments to prevent pneumonia are described in particular in the Annales Françaises d'Anesthésie et de Réanimation (30; 2011; 168-190). Conversely, a patient who does not present a risk of occurrence of a healthcare-associated infection can be quickly referred to a day hospital service, for example an infectiology service, rather than remaining in a service with close monitoring. which he won't need.

The invention will be described more precisely by means of the examples below. LEGENDS OF FIGURES

[Fig 1]: Representation of the genome structure of a TTV isolate.

[Fig 2A] [Fig 2B]: Characteristics of patients in the cohort at admission and results according to viral titer in TTV, kinetics of viremia and coinfection with herpesviruses (Figures 2A and 2B). Categorical variables are expressed in n (%) and continuous variables in median [Q1-Q3]. Comparisons between TTV conditions regarding titer or kinetics of viremia or herpesvirus coinfection were made using a Chi-square test for qualitative variables and a Wilcoxon test for variables quantitative, as appropriate. P values in bold with stars indicate significance at p < 0.05. ICU Intensive Care Unit, IAI ICU Acquired Infection, HpV Herpesvirus, SOFA Sequential Organ Failure Assessment, SAPS Simplified Acute Physiology Score, a TTV DNAemia less than 10,000 copies/ml, b TTV DNAemia greater than 10,000 copies/ml, c Stability of the TTV titer corresponding to a slope in the range -1.25/+1.25 (measured between the median viral titers at (D1 to D7) and (D14 to D28)), d Increase in TTV titer corresponding to a slope of >+1.5, e Decrease in TTV titer corresponding to a slope of <-1.5, f TTV DNAemia greater than 10,000 copies/ml and at least one herpesvirus, g Exclusive TTV DNAemia greater than 10,000 copies/mL, h exclusive herpesvirus (HpV). [Fig 3]: Association between the occurrence of HAI during the month of hospitalization and the presence of viremia.

[Fig 4]: Characteristics of patients in the cohort at admission and results according to TTV DNAemia greater than 167 copies (cp)/ml (LOD), 7000 copies/ml, 10,000 copies/ml and 40 000 copies/ml, during the first month (D1 to D28) following admission.

[Fig 5]: Association between binary clinical outcome and TTV expression kinetics.

[Fig 6]: Reference of HpV virus strains.

[Fig 7]: Reference table of PCR conditions for determining the LOD of viruses. EXAMPLES

In order to better understand the underlying pathophysiology of viral reactivation phenomena in patients admitted to the intensive care unit, a study was conducted on the cohort of patients involved in the REALISM clinical study (NCT01931956). For each of these patients, the blood DNAemia of the anellovirus TTV and of the herpesviruses CMV, EBV, HHV6, HSV-1 was measured over a period of one month following their admission to intensive care. The expected results aimed to identify common complications related to viral reactivation phenomena in these patients and to identify whether one or more viruses may be associated with the occurrence of specific complications.

Material and methods

Study population

An observational study was carried out on a prospective cohort of seriously ill patients who presented either for sepsis, severe trauma, severe burns or who had undergone planned surgery in the anesthesiology and intensive medicine department of the Edouard hospital. Herriot (Hospices Civils de Lyon, France). The inclusion period was 28 months (December 2015 - March 2018). The study protocol was approved by the institutional ethics committee (Comité de Protection des personnes Sud-Est II) under number 2015-42-2. This clinical study has also been registered on clinicaltrials.gov (NCT02638779). At the time of inclusion, written informed consent was obtained from each healthy volunteer and each patient. When a patient was unable to consent directly, informed consent was obtained from the patient's legal representative and reconfirmed with the patient at the earliest opportunity.

Inclusion criteria were: patients over 18 years of age, clinical diagnosis of sepsis as defined by the 2016 SEPSIS-3 consensus guidelines (Singer et al., 2016), severe trauma with a severity score (ISS) > 15, severe burn with total burn area greater than 30%, or surgical patients undergoing major surgery such as esophagogastrectomy, bladder resection with Brickers reconstruction, cephalic pancreaticoduodenectomy, and abdominal aortic aneurysm surgery by laparotomy. The exclusion criteria were as follows: presence of a pre-existing condition or of a treatment that could influence the immune status of the patients, in particular, patients on immunosuppressive treatment, pregnancy, institutionalized patients, inability to obtain informed consent are excluded.

At the same time, a cohort of 175 healthy volunteers aged 18 to 82 (81 men and 94 women) was prospectively recruited. To take into account the possible influence of age and sex on immune parameters, the distribution of healthy volunteers was based on age and sex demographic data of the French population in 2016.

Sampling and data collection

For each patient, samples and clinical data were collected three times during the first week after admission: (1) on day 1 or 2 (D1-2), (2) on D3 or 4 (D3- 4) and (3) on D5, D6 or D7 (D5-7) then twice more during the month following admission, (4) on D14 and (5) on D28. For healthy volunteers, a sample was taken during the study visit and clinical data was recorded. Data collection is detailed elsewhere (Venet et al., 2021).

Patient demographics, comorbidities, diagnosis, severity, and clinical outcomes were manually recorded prospectively. Longitudinal follow-up was carried out for a period of 30 days. Peripheral whole blood collected from each patient and from each healthy volunteer was processed each time within 3 hours of blood collection.

Blood was collected in one EDTA tube for determination of plasma viral ADNemia, immune phenotyping by flow cytometry and measurements of plasma cytokine levels, in two heparin tubes for functional tests (proliferation, cell production experiments cytokines), and finally in a PAXgene Blood RNA Tube (PreAnalytix, Hilden, Germany) for measurements of mRNA concentration of whole blood biomarkers by real-time PCR using Eva-Green or Taqman probe methodologies.

PAXgene samples were stabilized for at least 2 hours at room temperature after collection and then frozen at -80°C according to the manufacturer's recommendations. Complication Definitions

The main complications assessed in this study were secondary infection (HCA-associated infection) at 28 days (D28) and mortality at D28. Other complications assessed included intensive care unit (ICU) length of stay and total length of hospital stay, daily use of an invasive medical device (tracheal intubation, indwelling urinary catheter, and central venous line ). The information collected about the infections was reviewed by an independent decision-making committee made up of three clinicians not involved in the recruitment or care of the study patients.

The confirmation of the diagnosis of secondary infection (HCAI) by this committee was based on the guidelines of the European Society for Clinical Microbiology and Infectious Diseases and the American Society for Infectious Diseases. Only the first episode of secondary infection was taken into account in the analyses.

Determination of viral DNAemia

The semi-automated process for viral DNAemia determination of TTV and four herpesviruses, incorporating sample processing and standardized real-time quantitative PCR procedures, has previously been described in detail (Mallet et al., 2019 - Intensive Care Medicine Exp. 7:28).

Briefly, viral DNA extractions from plasma samples were performed using the Maxwell® HT Viral TNA chemistry kit (Promega) composed of paramagnetic silica particles and the Freedom EVO® liquid handling robot (TECAN) following the manufacturer's instructions. PCR controls were added to each plasma sample prior to extraction. The limit of detection (LOD - "Limit Of Detection") was previously determined for the semi-automated process as the smallest amount that can be distinguished from no detection (blank value) using viral standards (Mallet, 2019 - Intensive Care Medicine Exp. 7:28).

The detection limit of TTV was determined using plasma samples comprising a predetermined amount of TTV. First, the control plasma sample is serially diluted from 10 ¹ to 10 ⁴ , and each diluted sample is independently extracted 3 times and then amplified by quantitative PCR, in order to determine an approximate detection limit. In a second step, the greatest dilution giving a positive signal is diluted again to 1/2, and to 1/4 then each diluted sample is extracted independently 20 times and amplified. The most dilute positive replicates with 100% detection are used as the detection limit.

The LOD, expressed in copies per ml of plasma, is 167 for TTV, 100 for CMV, 33 for EBV, 166 for HHV6, and 33 for HSV1. Real-time PCR amplifications of viral DNAs were performed on the StepOnePlus™ Real-Time PCR System (ThermoFisher SCIENTIFIC) using R-GENE® assay kits using TaqMan probes for CMV, EBV , HSV1, HHV6 and TTV (bioMérieux SA). All nucleic acid samples, randomly distributed in the plates, were amplified simultaneously with the quantification standards, sensitivity controls and negative controls, according to the manufacturer's instructions.

Finally, using standard curves, the Ct (cycle threshold) values for each sample were converted to copies/pL of viral DNA in the PCR reaction tube and then to copies/mL viral DNA in plasma samples.

Statistical analysis

The viremia was evaluated by measuring the DNAemia for each virus, a viremic event, that is to say a positive DNAemia, being characterized by a number of copies of the virus per microliter greater than a predefined threshold (greater than the LOD). A positive DNAemia is considered early if it is detected for at least one of the patient's samples during the first week (day 1/2, day 3/4 or day 5/7) after the patient's admission to intensive care. DNAemia was also assessed on D14 and D28. Data are presented as numbers and percentages (qualitative variables) and medians and 25th/75th percentiles (quantitative variables).

The association between a viraemic event and clinical complications (survival or bacterial infection acquired in intensive care as previously described (Mallet et al., 2019 - Intensive Care Medicine Exp. 7:28) was assessed using the x2 test (chi-square or chi-square) or Fisher's exact test, if applicable.

Binary association between DNAemia/viremic event and quantitative immune markers (cells, cytokines, mRNA) was assessed using the the sum of Wilcoxon ranks. Finally, the analyzes were performed with R software version 3.4.4 and statistical significance was defined by an alpha risk of wrongly rejecting the null hypothesis of 5% (p < 0.05 indicates a significant association).

Results

Description of the cohort

The REALISM cohort of 377 critically ill patients consists of: 107 sepsis, 137 severe trauma, 24 severe burns and 109 major surgeries.

Quantitative (viral load) rather than qualitative (DNAemia) data may allow better discrimination between (1) insignificant viral load, (2) viral "reactivation" as a putative marker of immunosuppression, and (3) viral loads elevated supporting a true viral infection requiring treatment (Textoris et al., 2017).

Patients with high TTV DNAemia (viral load greater than 10,000 copies/mL) were more severely ill, with higher severity scores and increased need for treatment (Figure 3).

While the increase in viral load in TTV (p=0.025) was significantly correlated with a longer length of stay in intensive care, both the increase (p=0.048) and the decrease (p=0.048) in viral load in TTV, in other words a change in viral load in TTV, were significantly associated with the occurrence of an infection acquired in the intensive care unit during the first month (Figures 2A and 2B).

Moreover, it is surprising to find that cases of HAI tend to be more frequent in patients with positive DNAemia for TTV and at least one herpesvirus than in patients with only positive herpes DNAemia (Figures 2A and 2B) .

DNAemia in Intensive Care Unit Patients During the First Week and Month of Hospitalization - During the First Week

TTV was detected (positive DNAemia) in 217 (58%) of the patients in the cohort, slightly more than in the healthy controls (51%). TTV was detected alone in 160 (42%) patients, and co-detected in patients with 1 (n=48, 13%) or 2 (n=6, 2%), or 3 or more (n=3, 1 %) herpesvirus. The most frequently co-detected herpesvirus with TVT was EBV (n=29.8%), followed by HHV6 (n=18.5%), HSV1 (n=12.3%) and CMV (n=12.3%).

Overall, the number of patients with positive DNAemia for a single virus (n=189), whether herpesvirus or TTV, was similar to the number of patients with positive DNAemia for the presence of at least two viruses (n=188).

- During the first month

TTV was detected in 228 (60%) patients in the cohort. 151 (40%) patients had positive DNAemia for TTV only, while TTV was co-detected (positive DNAemias) in patients with 1 (n=59, 16%), 2 (n=11, 3%) or 3 or more (n=7.2%) herpesvirus. All herpesviruses were co-detected with TTV, the most frequent co-detection being with EBV (n=36.10%), then with HSV1 (n=28.7%), with CMV (n=23.6%) and with HHV6 (n=19.5%). Overall, the cohort included as many patients with an infection (positive DNAemia) for a single virus (n=187) as patients with a co-infection (n=190).

The percentage of patients with VTT was similar in patients with sepsis, severe trauma and major surgery (56-64%) and slightly lower in burn patients (46%), compared to the 51% seen in healthy volunteers .

In trend, the percentage of patients with a high titer of TTV seemed greater in the group of patients who underwent surgery and the severity criteria are correlated with a high viral load in TTV (especially greater than 7000 copies/mL) (Figure 4).

Association of DNAemia with clinical complications

Considering at the end of the 28-day observation period, the occurrence of at least one episode of healthcare-related secondary infection.

The detection of each herpesvirus individually during the first week of admission is not significantly associated with a risk of healthcare-associated infection (HAI), unlike the first month. A significant increase in the rate of IAS is observed in patients who have had at least one episode during the first month. viraemic (positive DNA) for any of the individual herpesviruses, in the absence of positive DNA for TTV.

Furthermore, a positive DNAemia for TTV alone (without detection of a herpesvirus) is associated with a reduced occurrence of IAS (Figure 3).

It was also observed that a stable TTV viral load during the first month is associated with a reduced occurrence of HAI (Figure 5).

If we consider the viral DNAemia of the patients of the cohort during the first month, we observe in patients presenting several viraemic events (DNAemia positive for 2 or more herpesviruses, or for a TTV and at least one herpesvirus) a rate higher HAI (44%) compared to patients with positive DNAemia for a single virus (22%) or patients without detected DNAemia (19%) (test x2. p <0.001).

Considering positive viral DNA events during the first week, patients who had multiple viraemic events had a similar HAI rate (11%) compared to those with positive DNA for only one. virus (9%) or no DNAemia detected (9%) (test x2. P = 0.8).

Surprisingly, the results show that early on, from the first week following the patient's admission, the co-detection of a high TTV viral load (in particular greater than 10,000 copies/ml of sample) and of a positive DNA for a herpesvirus is significantly associated with the risk of occurrence of a healthcare-associated infection (Figure 3).

To summarize, with respect to viremia, disease group and admission criteria, (i) the proportion of patients with variable (increasing or decreasing) TTV viral load over time tends to be higher in patients with sepsis than in other groups, and (ii) positive herpetic DNAemia as well as elevated TTV viral load appear to be associated with the severity of the patient's condition on admission.

With regard to HAIs, (i) co-detection, preferably from the first week of admission, of a high viral load in TTV with positive herpes DNAemia is correlated with the risk of more frequent occurrence of HAIs, and (ii) a stable TTV viral load is associated with relatively less occurrence of HAI, in contrast to a variation in the viral load in TTV which is also correlated with the risk of occurrence of IAS in the patient.

Discussion

The results demonstrated that it was possible to distinguish a non-significant TTV load from a high TTV load. Thus, it is to the credit of the inventors to have in particular identified a TTV expression threshold associated with physiopathological characteristics in patients in intensive care.

In brief, the results presented previously (i) demonstrated that the detection in the patient within the first week of ICU admission of a high load of TTV associated with herpes viral reactivation was associated with an increased risk of IAS, and that (ii) high early TTV viremia was associated with immune impairment.

The course of viremia over time was less discernible for TTV (from 58% in the first week to 60% over the month), and positive viremia for TTV was more common for TTV alone than for TTV co-detection -herpesvirus (42% against 15% measured during the first week, 40% against 20% measured during the first month).

Although the prevalence of TTV is higher in males in cohorts of healthy individuals (Focosi et al., 2020) and transplant patients (Bal et al., 2018), no gender bias was found. observed in the population of this study except for a male predominance in the subgroup with viral loads in TTV greater than 10,000 copies/mL.

TTV and EBV were the most frequently co-detected viruses, 8% to 10% during the first week and first month, respectively.

During the same period, only variations in the TTV burden are significantly associated with an increased risk of occurrence of IAS. The mere presence of TTV, including with a high load, is not in itself sufficient to determine the increased risk of IAS.

Nevertheless, during the first month after admission, patients with multiple viraemic events, including those with a positive DNAemia for TTV, have a higher frequency of HAI (44%) than those with having had one event. single viraemic or no viraemic event (22% and 19%, respectively). Thus, quite surprisingly and unexpectedly, the co-detection of a high load of TTV and a herpesvirus during the first week of admission is significantly associated with the occurrence of an IAS event during the first month.

In conclusion, the results demonstrate in patients admitted to the intensive care unit, the existence of a correlation between viral reactivation, in particular TTV in combination or not with HpV, and the risk of development of healthcare-associated infections.

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Claims

1. Method for in vitro or ex vivo evaluation of the risk of complication in a patient admitted to a health establishment and who is not under immunosuppressive treatment, characterized in that it comprises the implementation of the steps consisting of: a) measuring the viral load of at least one TTV in a biological sample from said patient, b) comparing the viral load determined for said biological sample with a predetermined reference value, and c) establishing a risk of complication when the viral load determined for said sample is higher or lower than the predetermined reference value.

2. Method according to claim 1, characterized in that the predetermined reference value is 10,000 copies/ml.

3. Method according to claim 2, characterized in that it is established that there is an increased risk of complication when said viral load determined for said sample is greater than said reference value.

4. Method according to claim 1 or 2, characterized in that it also comprises the detection of the presence of at least one Herpesvirus, preferably by measuring DNAemia.

5. Method according to claim 4, characterized in that it is established that there is an increased risk of complication when the viral load of at least one TTV determined for said sample is greater than 10,000 copies/ml and when at least one Herpesvirus is present.

6. Method according to any one of claims 1 to 5, characterized in that it comprises the implementation of the steps consisting in: a) measuring a first viral load of at least one TTV in said biological sample of the patient from of a first sample taken at time T1, b) measuring a second viral load of at least one TTV in said biological sample of the patient taken from a second sample taken at time T2, c) calculating the variation between the viral load at T2 and the viral load at T1, giving a ATTV value, d) drawing a conclusion on the risk of complication, based on the result of the comparisons.

7. Method according to claim 6, characterized in that it is concluded that there is no increased risk of complication when the ATTV value is within the range from -1.25 to +1.25, preferably from -1.20 to +1.20, and more preferably from -1.10 to +1.10.

8. Method according to any one of claims 1 to 7, characterized in that the risk of complication is the risk of occurrence of a healthcare-associated infection (IAS).

9. Method according to any one of claims 1 to 8, characterized in that the biological sample comes from a patient admitted to a hospital, preferably to the emergency department, the intensive care unit, in an intensive care unit or a continuing care unit.

10. Method according to any one of claims 1 to 9, characterized in that the biological sample comes from a patient in a septic state, more particularly, in septic shock, from a patient suffering from burns, more particularly from severe burns, a patient suffering from trauma, more particularly severe trauma, or a patient who has undergone a surgical operation, more particularly a major surgical operation.

11. Method according to any one of claims 1 to 10, characterized in that the biological sample comes from a patient in a septic state, suffering from burns, suffering from trauma or having undergone a surgical operation, and admitted to the unit intensive care.

12. Method according to claim 4 or 5, characterized in that said at least one Herpesvirus is selected from the group consisting of: CMV, EBV, HHV6 and HSV-1, and is preferably EBV.

13. Method according to any one of claims 1 to 12, characterized in that the biological sample is a biological fluid from the patient selected from the group consisting of blood or its derivatives such as plasma and/or serum, cerebrospinal fluid, urine, and bronchoalveolar lavages.