Use of Torque Teno Virus (TTV) as a marker to determine the risk of complications in patients admitted to health care facilities

Abstract

The present invention relates to a method for assessing the risk of complications in vitro or ex vivo in a patient admitted to a medical facility and not receiving immunosuppressive treatment, the method comprising measuring the viral load of at least one Torque Teno Virus (TTV) in a biological sample from the patient.

Description

[Technical field]

[0001] The present invention relates to the use of Torque Teno Virus (TTV) as a marker for determining the risk of complications in patients admitted to a medical facility. [Background technology]

[0002] Secondary infections (or healthcare-associated infections, HAIs) are a major complication of medical procedures, especially in healthcare facilities such as hospitals, and are referred to as nosocomial infections. Nosocomial infections occur in 20-40% of patients admitted to intensive care units, often in combination with the use of invasive medical devices and the administration of larger doses of antibiotics, resulting in longer hospital treatment. Finally, morbidity and mortality are also higher. The occurrence of healthcare-associated infections has become especially worse in the last few years due to the rise of multidrug-resistant pathogens. The World Health Organization (WHO) estimates that the number of nosocomial infections in European hospitals is about 5 million, leading to about 50,000 deaths and additional annual costs of 13 to 24 billion euros. Recommendations have been published and infection control programs encouraged, notably by the US Department of Health and Human Services, the European Centre for Disease Prevention and Control, the WHO and national agencies, where preventing and reducing healthcare-associated infections is a top priority. In one model, tests that reduce the time it takes to identify patients at high risk of acquiring healthcare-associated infections reduce mortality in these patients and are cost-effective. It is therefore important to be able to identify early markers associated with the risk of HAI, and more generally, the risk of developing a complication during a medical procedure, in order to provide an appropriate therapeutic response, particularly as soon as the risk is detected.

[0003] Numerous factors affect the occurrence and progression of healthcare-associated infections, including factors related to the patient's treatment as well as factors related to the patient's general health status. Some studies have demonstrated the existence of a relationship between a compromised immune system and an increased incidence of secondary infections (Non-Patent Document 1), but these results are controversial and more recent studies have questioned the existence of a relationship (Non-Patent Document 2). Some studies have also attempted to establish a link between the phenomenon of viral reactivation (especially herpesviruses (HPV) and torque teno virus (TTV)) and the occurrence of healthcare-associated complications. The phenomenon of viral reactivation is particularly present in patients who are not immunosuppressed, who have suffered severe illness (sepsis) or injury (trauma, burns, surgery), and in patients with immunoparalysis (characterized by the dysfunction of the adaptive and innate immune systems in response to an initial hyperinflammatory state).

[0004] Currently, the clinical significance of viral reactivation has not been clearly established. In particular, it is unclear whether reactivating viruses should be regarded as simple markers reflecting changes in the immune system or, on the contrary, as pathogens promoting secondary infections and requiring preventive measures (Non-Patent Document 3). Recently, Non-Patent Document 4 published the results of a study suggesting the existence of an association between co-reactivation of EBV (Epstein-Barr virus) and TTV virus and a non-significant reduction in mortality. Another recent study conducted in patients who received kidney transplants established that measuring TTV viral load can identify patients at low risk of secondary infection. (Non-Patent Document 5). Nevertheless, the scope of this study remains limited, and is even more limited because all the patients involved were on immunosuppressive treatment, which means they are at higher risk of developing infectious diseases. Therefore, it cannot establish a reliable association between TTV viral load and the risk of secondary infection (or healthcare-associated infection), nor can it identify which patients are at high risk of developing secondary infection. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Focosi D, Antonelli G, Pistello M, Maggi F. Torquetenovirus: the human virome from bench to bedside. Clin Microbiol Infect. July 2016;22(7):589-93. [Non-Patent Document 2] J. Venet et al., 2021 [Non-Patent Document 3] J. Limaye et al., 2010 [Non-Patent Document 4] Mallet et al.,2019-Intensive Care Medicine Exp.7:28 [Non-Patent Document 5] Strassl et al.,The Journal of Infectious Diseases,2018;218:1191-9 Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there remains a need for simple and reliable diagnostic methods that can identify patients at risk of developing complications, especially healthcare associated infections, while undergoing medical procedures in a healthcare facility. [Means for solving the problem]

[0007] A first subject of the present invention relates to a method for assessing, in vitro or ex vivo, the risk of complications in a patient admitted to a medical facility and not receiving immunosuppressive treatment, comprising measuring the viral load of at least one Torque Teno Virus (TTV) in a biological sample from said patient. In a preferred embodiment, the method involves a patient admitted to a hospital, preferably an emergency department, resuscitation department, intensive care unit or high dependency facility. In another preferred embodiment, the method is directed to a patient in which the patient is in a septic state, more particularly a patient in septic shock, a patient suffering from burns, more particularly a patient suffering from severe burns, a patient suffering from trauma, more particularly a patient suffering from severe trauma, or a patient undergoing surgery, more particularly a patient undergoing major surgery. In another preferred embodiment, the method is such that the biological sample is derived from a patient who is in a septic state, who has suffered a burn, who has suffered a trauma, or who has undergone surgery and who has been admitted to an intensive care unit. In another preferred embodiment, the method comprises the step of: the complication is the occurrence of a healthcare-associated infection (HAI).

[0008] In another preferred embodiment, the method comprises the steps of: a) determining the viral load of at least one Torque Teno Virus (TTV) in a biological sample from said patient; b) comparing the viral load determined for the biological sample with a predetermined reference value; and c) establishing the risk of complications if the viral load determined for the biological sample is greater or less than the predetermined reference value; This includes performing the following: In another preferred embodiment, the method comprises the step of determining whether the predetermined reference value is 10,000 copies / ml.

[0009] In another preferred embodiment, the method comprises the steps of: a) measuring a first viral load of at least one TTV in a biological sample of said patient derived from a first sample taken at time T1; b) measuring a second viral load of at least one TTV in a biological sample of said patient derived from a second sample taken at said time T2; c) calculating the variation between the viral load at T2 and the viral load at T1 to obtain a ΔTTV value; and d) drawing conclusions about the risk of complications from the results of the comparison; This includes performing the following: In a preferred embodiment, the method comprises the steps of: a) measuring a first viral load of at least one TTV in a biological sample of said patient derived from a first sample taken at time T1; b) measuring a second viral load of at least one TTV in a biological sample of said patient derived from a second sample taken at said time T2; c) calculating the variation between the viral load at T2 and the viral load at T1 to obtain a ΔTTV value; and d) Draw the conclusion that there is no increased risk of complications when the ΔTTV value is within the ranges of -1.25 to +1.25, -1.20 to +1.20, or -1.10 to +1.10; This includes performing the following: In another preferred embodiment, the method further comprises detecting the presence of at least one herpes virus. In another preferred embodiment, the method also comprises determining the viral load of at least one herpes virus (HPV). In another preferred embodiment, said at least one herpes virus (HPV) is selected from the group consisting of CMV, EBV, HHV6 and HSV-1, preferably EBV. In another preferred embodiment, the method measures the viral load by amplification, sequencing or hybridization, preferably by amplification, more preferentially by real-time PCR, of at least one TTV sequence and, where appropriate, at least one herpesvirus.

[0010] In another preferred embodiment, the method comprises the step of: the biological sample is a biological fluid from a patient, said biological fluid being selected from the group consisting of blood or a derivative thereof, plasma and / or serum, cerebrospinal fluid, urine, and bronchoalveolar lavage fluid. [Brief description of the drawings]

[0011] [Figure 1] Representation of the genomic structure of TTV isolates. [Diagram 2]Patient characteristics from the cohort at admission and outcomes based on TTV viral titers, viremia dynamics and concomitant infection with herpesvirus (Figures 2A and 2B). Categorical variables are expressed as n (%) and continuous variables as median [Q1-Q3]. Comparisons between TTV conditions regarding titers or viremia dynamics or coinfection with herpesvirus were performed using chi-squared tests for qualitative variables and Wilcoxon tests for quantitative variables, as appropriate. Bold P values ​​with asterisks represent significance at p<0.05. ICU=intensive care unit, HAI=ICU-acquired infection, HPV=herpesvirus, SOFA=sequential organ failure assessment, SAPS=simplified acute physiology score. a TTV DNAemia below 10000 copies / ml, b TTV DNAemia above 10000 copies / ml, c Stability of TTV titer corresponding to a slope within the range of -1.25 / +1.25 (measured between the median viral titers at (D1–D7) and (D14–D28)), d Increase in TTV titer corresponding to a slope >+1.5, e Decrease in TTV titer corresponding to a slope <-1.5, f TTV DNAemia above 10000 copies / ml and at least one herpes virus, g Only TTV DNAemia above 10000 copies / ml, h Only herpes virus (HPV). [Diagram 3] Association between occurrence of HAI and presence of viremia during month of hospitalization. [Figure 4] Characteristics of patients of the cohort at admission and outcomes based on TTV DNAemia of >167 copies (cp) / ml (LOD), >7000 copies / ml, >10000 copies / ml and >40000 copies / ml during the first month after admission (D1–D28). [Diagram 5] Association between binary clinical outcomes and the dynamics of TTV expression. [Figure 6] HPV virus strain references. [Figure 7] Reference table of PCR conditions for determining viral LOD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The details will be explained below. The subject of the present invention is a method for assessing the risk of complications, such as healthcare-associated infections (HAI), in patients admitted to a healthcare facility and not receiving immunosuppressive treatment. Indeed, the inventors have shown that Torque Teno Virus (TTV) viral load is an indicator of the risk of complications in patients not receiving immunosuppressive treatment and admitted to a medical facility, more specifically in patients treated in intensive care units. Notably and surprisingly, the increase in TTV viral load after the first week after admission to intensive care appears to correlate significantly with a longer stay in the unit. Furthermore, both increases and decreases in TTV viral load are significantly associated with the occurrence of HAI during the first month after hospitalization. Finally, the simultaneous detection of a high TTV viral load associated with the presence of at least one herpesvirus during a patient's hospitalization, especially from the first week of hospitalization in the intensive care unit, correlates with the risk of developing a healthcare-associated infection (HAI). Thus, an early assessment of the risk of developing complications, especially of developing a healthcare-associated infection (HAI), can be performed in patients not receiving immunosuppressive treatment. In this specification, the embodiments may be implemented alone or in appropriate combination.

[0013] Methods for assessing a patient's risk of complications The subject matter of the present specification is a method for assessing the risk of complications in a patient admitted to a medical facility and not receiving immunosuppressive treatment based on the viral load of at least one Torque Teno Virus (TTV) in a biological sample from the patient, in vitro or ex vivo. In a first aspect, a method for assessing the risk of complications in a patient admitted to a medical facility, in vitro or ex vivo, is described herein, comprising measuring the viral load of at least one Torque Teno Virus (TTV) in a biological sample from the patient, characterized in that the patient is not receiving immunosuppressive treatment. Surprisingly, by studying the phenomenon of viral reactivation in patients in a health care facility, the inventors identified the existence of a correlation between the change in the TTV viral load during the stay in a health care facility in patients not receiving immunosuppressive treatment and the occurrence of complications, i.e. unfavorable changes in the patient's condition, i.e. in particular the occurrence of health care-associated infections. In a preferred embodiment, the variation in TTV viral load during the first month after admission to a health care facility is indicative of the risk of developing an HAI.

[0014] As used herein, the term "assessing a patient's risk of a complication" or "determining a patient's risk of a complication" refers to determining the probability that a subject or patient will develop a complication, as defined below, in the future. The methods disclosed herein represent tools for assessing said risk and can be combined with other processes, methods or indicators such as clinical testing, taking of a biological sample from said patient, and detection of one or more biomarkers and / or one or more infectious agents, in particular bacteria, viruses, parasites, yeasts or mold fungi. As used herein, "complication" refers to an unfavorable change in a disease, condition, or medical treatment. In this context, "complication" may refer specifically to an unfavorable change in a disease that may require the implementation of one or more invasive medical devices, such as the insertion of an endotracheal intubation, a urinary catheter, a catheter, or a central line. "Complications" also refers to unfavorable developments of a disease that may result in prolonged treatment in the medical facility, especially in an intensive care unit. "Complication" also means death of the patient. "Complication" preferably refers to an unfavorable change in the disease that may require a particular medical procedure to be performed. Examples of such complications include secondary or opportunistic infections, particularly the development of "healthcare associated infections" or "HAIs", such as infections caused by one or more infectious agents, e.g., bacteria, viruses, parasites, yeasts or molds.

[0015] In the context of this specification, an infection is said to be "healthcare-associated infection" if it occurs during the treatment of a patient by a healthcare professional (diagnostic, therapeutic, palliative, preventive, educational or surgical) and if it was not present at the start of treatment. Healthcare-associated infection encompasses infections acquired in a healthcare facility (referred to as nosocomial infections) as well as infections acquired during care received outside this setting. If the status of the infection is not precisely known at the start of treatment, a delay of at least 48 hours or longer than the incubation period is generally acceptable to define an HAI. For infections at the surgical site, infections occurring within 30 days after surgery or, if an implant, prosthesis or prosthetic material is in place, within one year after surgery are considered to be healthcare-associated infections. Healthcare-associated infections can be of bacterial origin, for example with bacteria such as Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa. Infections can also be of fungal origin, in particular with yeasts of the genus Candida or with filamentous fungi of the genus Aspergillus, in particular Aspergillus fumigatus. Infections can also be viral, in particular with adenoviruses, herpesviruses, enteroviruses, rotaviruses or viruses of the HIV type. It can also be the reactivation of potentially pathogenic latent viruses, such as herpesviruses, for example the viruses CMV, EBV, HHV6 and HSV-1.

[0016] In a preferred embodiment, the present disclosure relates to a method for in vitro or ex vivo assessment of the risk of a complication in an immunosuppressively untreated patient, the complication being the development of a healthcare-associated infection (HAI). In particular, the HAI may be a hospital-acquired infection. In this case, the method described herein can determine the risk of developing a hospital-acquired infection in the patient. In the case of a patient who is in a septic state, and therefore already suffers from a primary infection, the method described herein can determine the risk of developing a secondary infection.

[0017] The methods described herein are in particular based 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 obtained from a subject. In general, a biological sample should allow the determination of TTV and / or herpes virus (HPV) load, where biological sample means, inter alia, but is not limited to, whole blood, serum, plasma, sputum, nasopharyngeal sample, urine, stool, skin, cerebrospinal fluid, saliva, gastric secretions, sperm, semen, tears, spinal tissue or fluid, cerebrospinal fluid, trigeminal ganglion sample, sacral ganglion sample, adipose tissue, lymphatic tissue, placental tissue, upper genital tract tissue, gastrointestinal tract tissue, reproductive tissue and nervous system tissue. In particular, the sample may be a biological fluid such as a blood sample or a blood-derived sample, in particular selected from whole blood (taken from a vein, i.e. containing white and red blood cells, platelets and plasma), plasma, serum, and all types of cells extracted from blood, such as peripheral blood mononuclear cells (PBMCs, including lymphocytes (B, T and NK cells), dendritic cells and monocytes), B-cell subpopulations, purified monocytes or neutrophils. The test sample may be used directly from a biological source or after a pretreatment that may modify the properties of the sample. For example, such pretreatment may include preparation of plasma from blood, dilution of viscous fluids, etc. Pretreatment methods may also include filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of disruptive components, addition of reagents, dissolution, 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 of the patient, said fluid being selected from the group consisting of blood or a derivative thereof, such as plasma and / or serum, cerebrospinal fluid, urine and bronchoalveolar lavage fluid. Even more preferably, the biological sample is a sample of blood or a derivative thereof, such as plasma and / or serum.

[0018] As used herein, the term "subject" refers to a vertebrate, preferably a mammal, and most preferably all humans. A human subject may be a "patient." "Patient" more specifically refers herein to a human subject in contact with a medical professional such as a physician (e.g., a general practitioner) or a medical or health facility (e.g., a hospital, more specifically, an emergency department, a resuscitation department, an intensive care unit or a high dependency facility, or an assisted living type of geriatric care facility). In certain embodiments, the patient may be an elderly person in the context of a vaccination protocol (particularly in a care home or a general practitioner). As described herein, the assessment method determines the risk of a complication in a patient not receiving immunosuppressive treatment. By "immunosuppressively naive patient" is meant that the patient is not taking any immunosuppressant drugs or medications that have immunosuppressive side effects and is not receiving any treatment that may induce immunosuppression. "Immunosuppressive treatment" or "inducing immunosuppression" refers to treatments, especially medicines, whose effect is to reduce, inhibit or prevent the activity of the immune system. As non-limiting examples, "immunosuppressive treatment" may be treatments that implement, in particular, glucocorticoids, cytostatics, interferons, antiproliferative drugs and antimetabolites (rapamycin, everolimus, mycophenolate mofetil, mycophenolic acid), calcineurin inhibitors, especially those for their anti-transplant rejection action (cyclosporine, tacrolimus (FK506), voclosporin), S1P-R agonists (FTY720), malononitrilamide (FK778), antibodies, for example antithymocyte globulin or certain monoclonal antibodies against specific antigens, such as muromonab-CD3, daclizumab, basiliximab, rituximab, alemtuzumab, infliximab, adalimumab, efalizumab, etc. "Immunosuppressive treatment" also means radiation therapy or chemotherapy. In a preferred embodiment, the present disclosure relates to a method for assessing, in vitro or ex vivo, the risk of complications in patients not receiving immunosuppressive treatment and admitted to a healthcare facility, in particular an emergency department, a resuscitation department, an intensive care unit or a high dependency facility. A distinction is made between certain departments within a hospital depending on the medical situation of the patient. "Emergency Department" means a department that receives sick and injured people who have either arrived on their own or been transported by ambulance or emergency management organization vehicle. The function of the emergency management organization is to receive, without discrimination, any person in an emergency, including a psychiatric emergency, at all times of the day or night and throughout the year, and to treat such persons, particularly in cases of distress and life-threatening emergencies. "Resuscitation department" means a specialized department where the most critically ill patients are admitted. In this department, patients benefit from constant monitoring of vital functions such as ventilation, oxygenation, blood pressure, cardiac and renal function. If necessary, these vital functions can be supported in order to allow the patient to survive if possible. Patients are admitted to the resuscitation department when they show a failure of vital functions, for example during severe infections (septic shock), drug poisoning, multiple trauma, coma, acute renal failure, acute respiratory failure, after cardiac arrest or in the postoperative period after major operations such as cardiac or gastrointestinal surgery. "Intensive care unit", "Intensive care unit department" or "ICU" means a unit responsible for treating patients in a critical condition, i.e. those who exhibit a malfunction of at least one vital function or who are at risk of developing serious complications. Intensive care units have highly specialized technical means that are continuously used by a multidisciplinary team to detect, prevent and correct acute imbalances that are considered reversible and that are linked to underlying disorders (diseases, surgery, trauma, poisoning, burns, sepsis). "High Dependency Unit" or "HDU" means a hospital department for receiving and treating sick people who require close monitoring. High dependency units treat patients whose condition and treatment suggest that an impairment of one or more of their vital functions may occur, and whose condition after recovery from an impairment of one or more of their vital functions (e.g., after a stay in a resuscitation department) is too severe or unstable to allow return to a traditional hospital unit. High dependency units are an intermediate level between a resuscitation department and a traditional care department.

[0019] In a preferred embodiment, the in vitro or ex vivo assessment method of the present invention allows for the assessment of the risk of complications in patients admitted to an intensive care unit. The present assessment method is particularly suitable for assessing the risk of complications in patients suffering from an immunoinflammatory attack. "Immunoinflammatory insult" refers to a trauma or wound, the effect of which is to induce an exaggerated inflammatory response in the patient's body. The persistence of this exaggerated inflammatory response leads to acquired immunosuppression associated with an anti-inflammatory profile, which ultimately leads to unstoppable infection. This phenomenon, also known as immunoparalysis, is characterized by the downregulation of the expression of molecules of the major histocompatibility complex class II on the surface of monocytes but not on the surface of B cells. Suppression of monocyte / macrophage function reduces the release of immune complexes, negatively affects antigen-presenting capacity, and reduces the function of natural killer (NK) cells. This type of reaction is common in non-immunosuppressed patients in intensive care units (C. Hotchkiss et al., 2013). "Immunoinflammatory attack" particularly includes trauma, for patients suffering from trauma, burns, for patients suffering from burns, surgery, for patients who have undergone surgery, or sepsis, for patients in a septic state. In a preferred embodiment, the present description relates to a method for assessing, in vitro or ex vivo, the risk of complications in patients not receiving immunosuppressive treatment, in patients in a septic state, more particularly in septic shock, and / or in patients suffering from burns, more particularly in severe burns, and / or in patients suffering from trauma, more particularly in severe trauma, and / or in patients undergoing surgery, more particularly in major surgery. By "septic patient" or "patient suffering from sepsis" is meant a patient exhibiting at least one life-threatening organ failure caused by an inappropriate host response to infection. "Septic shock" refers to a subtype of sepsis in which hypotension persists despite adequate vascular filling. By "patient suffering from a burn" is meant that the patient has destruction of skin cells and underlying tissue caused by thermal and / or electrical and / or chemical and / or radiation burns. The burns may be superficial, partial or full thickness and may be generally or specifically located, for example, on the neck, face, eyes, hands, feet, joints or other parts of the body. By "severe burn" or "patient suffering from severe burn" is meant a patient suffering from a burn covering a body surface area of ​​more than 15% of the total body surface area, preferably more than 20%, preferably more than 25%, and most particularly more than 30% of the total body surface area. "Patient suffering from trauma" means a patient directly admitted to an intensive care unit. "Patient suffering from severe trauma" means a patient with an Injury Severity Score (ISS, Baker et al., 1974) of greater than 15, preferably greater than 20, more preferably greater than 25. "Surgical patient" means a patient who has undergone invasive surgery for the purpose of treating a disease state, such as a disease or wound, to assist or improve bodily function, appearance, or to repair a wounded area. "Major surgery" or "patient undergoing major surgery" refers to a surgical procedure that is technically difficult and / or involves a risk of bleeding and / or a risk of mortality and / or is prolonged (e.g., more than 3 hours, preferably more than 4 hours, e.g., 5 or 6 hours, or even longer) and / or requires significant post-operative care. By way of non-limiting example, major surgery refers to surgery intended for at least one of the following indications: esophagogastroctomy, Bricker's cystectomy (total cystectomy with reconstruction from small intestinal mucosa), head pancreaticoduodenectomy (Whipple procedure) and / or abdominal aortic aneurysm surgery by laparotomy.

[0020] Certain preferred embodiments are described below. The present specification is not limited to these embodiments, and other specific embodiments can be implemented by combining one or more of the above-mentioned features. Therefore, the inventors studied the phenomenon of viral reactivation in patients in a medical facility. More specifically, they measured the presence and / or variation of TTV viral load in biological samples from said patients at different time points of their treatment in a medical facility. The inventors have observed that the viral load may vary over time, particularly depending on the patient. In particular, the inventors have surprisingly observed that this variation indicates the risk of developing a healthcare-associated infection and / or the risk of prolonging the duration of treatment. Therefore, the inventors have developed a method for assessing the risk of complications in patients admitted to a healthcare facility in vitro or ex vivo. The methods described herein are based on the measurement of TTV viral load. "Torque Teno Virus" or "TTV" as used herein refers to a virus of the Anelloviridae family. TTV as intended herein is a non-enveloped virus with a circular, single-stranded DNA genome of negative polarity. "TTV genome" refers herein to any genome of any Anelloviridae family, in particular the genomes of the genera Torque Teno virus (TTV), Beta Torque virus (TTMV), and Gamma Torque virus (TTMDV) found in humans. By way of example, reference is made herein to the genome of the prototype Torque Teno virus strain TTV-1a. More specifically, an example of a TTV genome is the sequence represented, for example, by SEQ ID NO: 1, with Genbank accession number AB017610. In 1997, using gene subtraction techniques, a viral sequence fragment of approximately 500 base pairs (bp) was discovered in the serum of a Japanese patient presenting with posttransfusion hepatitis of uncertain etiology (1-7). This clone, initially designated N22 and, although not listed in any 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 studies subsequently showed that the TTV genome consists of circular single-stranded DNA of negative polarity. It was the first virus of circular single-stranded DNA isolated in humans. Using primers capable of amplifying sequences in this noncoding region, a high prevalence of TTV in the world population (approximately 90%) was demonstrated. TTV causes chronic infection without any obvious associated clinical signs. It is called a non-pathogenic or orphan virus. Therefore, many studies have focused on the involvement of TTV in human diseases, especially in certain liver diseases, but the function of this virus has not been clearly identified. Preferentially, the genome size of TTV is about 3.8 kb. The genome structure and organization of TTV are known to those skilled in the art (see, for example, Biagini, Curr Top Microbiol Immunol. 331:21-33, 2009) and are illustrated in FIG. Thus, the TTV genome can be divided into an untranslated region (UTR) of about 1-1.2 kb and a potential coding region of about 2.6-2.8 kb. The coding region contains, inter alia, two large open reading frames: ORF1 and ORF2, which code for 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. There may be other open reading frames in the TTV genome. For example, the genome of TTV may contain two further open reading frames, ORF3 and ORF4 (Figure 1). The untranslated region UTR is highly conserved. It contains, in particular, GC-rich sequences that are prone to forming secondary structures. Amplification of selected sequences in the UTR-5' untranslated region has demonstrated that the prevalence of the virus is very high throughout the entire population worldwide (Hu et al., J Clin Microbiol. 43(8):3747-3754, 2005). This region contains, in particular, a 128bp sequence, which can be amplified using methods known to those skilled in the art, for example, the TTV R-GENE® diagnostic kit (bioMerieux, France).

[0021] "Viral load" refers to the number of viral particles present in a biological sample. Viral load reflects the severity of viral infection. Viral load can be determined by measuring the amount of one of the components of the virus (genomic DNA, mRNA, protein, etc.) in a biological sample. Thus, preferably, the viral load 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 this case, viral load refers to TTV load. "TTV load" in this specification corresponds more specifically to TTV viral load, i.e. the number of particles of TTV virus present in biological sample. The TTV load of a patient means any TTV viral load that the patient carries. The TTV load can be determined by measuring the amount of TTV components in this biological sample. Preferably, TTV amount corresponds to the amount of TTV nucleic acid sequence present in biological sample.Therefore, determining the TTV amount of a patient according to the protocol of the present invention comprises estimating the number of all TTV sequences in the biological sample of said patient.In particular, the method of the present invention does not select the specific TTV strain measured in said biological sample.Indeed, the detection of high TTV viral load, especially the detection of fluctuations in TTV load, is associated with an increased risk of developing complications, especially healthcare-associated infection, regardless of the one or more Anelloviridae strains detected. Preferably, determining the amount of TTV comprises determining the amount of active and / or inactive viral copies, that is, determining the amount of circulating, integrated or latent viral copies. In particular, the TTV viral copy number can be determined by predetermining the TTV detection limit.

[0022] "Limit of detection" or "LOD" means the minimum amount of genome copies that can be distinguished from the absence of detection (blank value) using a viral standard. The amount of virus can then be determined in terms of copies of viral DNA per μl in the reaction tube and then in terms of copies of viral DNA per ml in the sample by extrapolation from the standard curve. In the context of this specification, a viral load in a biological sample of more than 7000 copies per ml of biological sample, e.g. blood or derivatives thereof such as plasma and / or serum, preferably more than 10000 copies per ml of plasma, preferably more than 20000 copies per ml of biological sample, more preferably more than 40000 copies per ml of biological sample, is considered to be a high TTV viral load. The TTV level, and therefore the amount of TTV, can be determined by measuring the level of TTV DNA, TTV RNA or TTV protein using methods well known to those skilled in the art. The method according to the invention may therefore comprise, between taking the sample from the patient and step a) defined below, other preliminary steps corresponding to converting the biological sample into a double-stranded DNA sample, or into an mRNA (or corresponding cDNA) sample, or into a protein sample, which is then ready to be used for the in vitro measurement of the TTV viral load in step a) defined below. The preparation or extraction of viral double-stranded DNA, mRNA (and its reverse transcription to obtain cDNA) or proteins from cell samples is simply a routine procedure known to those skilled in the art.

[0023] The double-stranded DNA can correspond to the entire genome of TTV or just a portion thereof. Once double-stranded DNA, mRNA (or corresponding cDNA) or ready-to-use protein sample is available, detection of TTV can be performed using genomic DNA (i.e., based on the presence of at least one sequence consisting of at least a portion of the TTV genome), using mRNA (i.e., based on the content of TTV mRNA in the sample), or using protein (i.e., based on the content of TTV protein in the sample), depending on the type or conversion of the sample. Preferably, TTV levels are determined by measuring levels of TTV nucleic acid, more preferably TTV DNA. Methods for detecting nucleic acids in a biological sample are well known to those skilled in the art and include, inter alia, amplification, preferably by PCR, more preferably by real-time PCR, sequencing, and hybridization with labeled probes. Advantageously, the present document therefore relates to a method for in vitro or ex vivo assessment of the risk of complications in a patient admitted to a medical facility and not receiving immunosuppressive treatment, comprising measuring the viral load of at least one Torque Teno Virus (TTV) in a biological sample of said patient, said viral load being measured by amplification, sequencing or hybridization of at least one TTV sequence, preferably by amplification and more preferentially by real-time PCR.

[0024] In a first embodiment, the amount of TTV is determined by amplification of the TTV sequence. In this embodiment, a preferred approach consists in amplifying a sequence known to be specific to the TTV genome, particularly preferably at least one sequence from the untranslated region (UTR) of TTV as defined above. "Sequence specific to the genome of TTV" means herein a sequence that is present in most of the known TTVs but not in most of the other viruses, in particular in most of the other anelloviruses. Preferably, the 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 the known TTV genomes. More preferably, it is present in 100% of the known TTV genomes. Alternatively, the 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 the known genomes of anelloviruses other than TTV. Preferably, the TTV-specific sequence is not present in all the known genomes of anelloviruses other than TTV. Particularly preferably, such a sequence is, for example, a sequence contained in an untranslated region (UTR) that is highly conserved from one TTV to another TTV. More specifically, such a sequence may correspond to a 128 bp sequence of the 5'-UTR untranslated region, which may be amplified by methods known to those skilled in the art, for example using the TTV R-GENE® diagnostic kit (bioMerieux, France). Thus, in this embodiment, the method described herein involves the use of primers and probes for the amplification of sequences known to be specific for the genome of TTV. As is customary in the art, the primers are preferably oligonucleotides. For example, the primers may contain less than 30 nucleotides, less than 25 nucleotides, less than 20 nucleotides, less than 15 nucleotides, or less than 12 nucleotides. Alternatively, these primers contain at least 12, 15, 20, 25 or 30 nucleotides. Preferably, the primers used contain 12-20 nucleotides, 12-25 nucleotides, 15-20 nucleotides, or 15-25 nucleotides. The skilled artisan knows how to determine the length and sequence of primers for the amplification of TTV-specific sequences. They may, for example, use the same primers as those provided in the TTV R-GENE® diagnostic kit (references 423414 and 69-030B, bioMerieux, France).

[0025] Amplification techniques include, in particular, isothermal techniques and techniques based on PCR (polymerase chain reaction). Isothermal amplification methods include a large number of methods. The most commonly used for pathogen detection are LAMP (loop-mediated amplification) and RPA (recombinase polymerase amplification) methods. Isothermal amplification methods also include, 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), ICAN (isothermal and chimeric primer-initiated amplification of nucleic acids), SMART (signal-mediated amplification of RNA technology) and other methods (e.g. Asiello et Baeumner, Lab Chip 11(8):1420-1430,2011). Preferably, the PCR technique used quantitatively measures the initial amount of DNA, cDNA or RNA. Examples of PCR-based techniques that can be used in the methods described herein include, but are not limited to, techniques such as real-time PCR (Q-PCR), reverse transcription PCR (RT-PCR), multiplex reverse transcription PCR, real-time reverse transcription PCR (QRT-PCR) and digital PCR. These techniques are well known and readily available to those skilled in the art. Preferably, the amount of TTV is determined using real-time quantitative PCR. Numerous methods for detecting and quantifying TTV have been reported in the art (e.g., Maggi et al., J Virol. 77(4):2418-2425, 2003). In particular, reference is made to the method described in the following document: 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 contained in the non-coding UTR region. This sequence is present in all known TTVs, thereby providing this method with a very high degree of specificity. Moreover, it is particularly versatile and can be carried out using any type of PCR platform. To carry out this method, it is particularly advantageous to use the TTV R-GENE® diagnostic kit (reference numbers 423414 and 69-030B bioMerieux, France).

[0026] Alternatively, viral load is determined using digital PCR. Digital PCR involves a PCR analysis on extremely diluted nucleic acids, so that the majority of positive amplifications reflect the signal from a single template molecule. Thus, digital PCR allows individual model molecules to be counted. The ratio of positive amplifications to the total number of PCRs analyzed allows the concentration of the template in the original or undiluted sample to be estimated. This technology has been proposed to detect various genetic phenomena (Vogelstein et al., Proc Natl Acad Sci USA 96:9236-924, 1999). Digital PCR, like real-time PCR, can potentially distinguish very small quantitative differences in target sequences between samples.

[0027] In another embodiment, TTV DNA levels are measured by sequencing. The term "sequencing" as used herein is given its broadest accepted meaning and refers to any technique known to those of skill in the art that allows for determining the sequence of a polynucleotide molecule (DNA or RNA), i.e., the sequence of the nucleotides that make up this molecule. Thus, the TTV DNA can be sequenced by any technique known in the art. Sequencing as meant herein includes, inter alia, Sanger sequencing, whole genome sequencing, sequencing by hybridization, pyrosequencing (especially 454 sequencing, Solexa Genome Analyzer sequencing), sequencing by capillary electrophoresis, cycle sequencing, single base extension sequencing, solid-phase sequencing, high-throughput sequencing, massively parallel signature sequencing (MPSS), reversible dye terminator sequencing, mate pair sequencing, short read sequencing, exonuclease sequencing, sequencing by ligation, single molecule sequencing, sequencing by synthesis, electron microscopy sequencing, real-time sequencing, inversion stop sequencing, nanopore sequencing, reversible terminator sequencing, semiconductor sequencing, SOLiD® sequencing, SMRT (single molecule real-time analysis) sequencing, MS-PET sequencing, mass spectrometry, and combinations thereof. Particular embodiments include, for example, the platforms MiSeq, NextSeq 500, and Illumina (Reuter et al., Mol Cell, 58:586-597, 2015; Bentley et al. Nature; 456:53-59, 2008), 454 genome sequencers and Roche (Margulies et al. Nature; 437:376-380, 2005), Applied Biosystems (McKernan et al., Genome Res; 19:1527-1541, 2009), the Polanator platform (Shendure et al., Science, 309:1728-1732), or the Helicos single molecule sequencing platform (Harris et al. Science; 320:106-109, 2008).High throughput sequencing also includes SMRT real-time sequencing (Roads et al., Genomics, Proteomics & Bioinformatics, 13(5):278-289, 2015), Ion Torrent sequencing (WO2010 / 008480); Rothberg et al., Nature, 475:348-352, 2011) and nanopore sequencing (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 a portion of the DNA contained in the biological sample. It is immediately clear to those skilled in the art that the sample contains at least a mixture of TTV DNA and host subject DNA. Moreover, TTV DNA probably represents only a small fraction of the total DNA present in the sample. Advantageously, DNA is randomly fragmented prior to sequencing, typically using physical methods. The first approach consists in sequencing specific sequences of the genome of the TTV species. Another approach consists in using methods that allow quantitative genotyping of nucleic acids obtained from biological samples with high accuracy. In certain embodiments, accuracy is obtained by analyzing a large number (e.g., millions or billions) of nucleic acid molecules without amplification, using protocols based on prior knowledge of the target sequence (i.e., in this case, the TTV sequence).

[0028] In a preferred embodiment, the methods of the invention include quantifying the number of reads. In certain embodiments, a random subset of nucleic acid molecules from biological samples is subjected to high-throughput sequencing.Preferably, TTV sequence is identified in the overall sequencing data by comparison with publicly deposited TTV sequence.This comparison is advantageously based on the level of sequence identity with known TTV sequence, and can even detect far-away variants.Currently available software such as BLAST can be used to determine the level of identity between sequences. Thus, sequences that are 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% identical to a known TTV sequence are identified as TTV sequences, In this embodiment, determining the amount of TTV thus comprises numbering the TTV sequences identified by sequencing in the biological sample from the subject.

[0029] In another embodiment, the amount of TTV is determined by measuring the amount of viral proteins in a biological sample, thus using specific antibodies in well-known techniques such as immunoprecipitation, immunohistology, Western blot, dot blot, ELISA or ELISPOR, electrochemiluminescence (ECLIA), protein chip, antibody chip, or tissue chip combined with immunohistochemistry, among others. Other techniques that may be used include FRET or BRET techniques, microscopy or histochemistry, including in particular confocal and electron microscopy, methods based on the use of one or more excitation wavelengths and suitable optical methods (e.g. electrochemical methods (voltammetric and amperometric techniques), atomic force microscopy, and radio frequency methods (e.g. multipolar resonance spectroscopy, confocal and non-confocal), detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance and birefringence or refractive index (e.g. surface plasmon resonance, ellipsometry, resonant mirror methods, etc.), flow cytometry, radioisotope or magnetic resonance imaging, analysis by polyacrylamide gel electrophoresis (SDS-PAGE), analysis by mass spectrometry and liquid chromatography coupled with mass spectrometry (LC-MS / MS). All these techniques are well known to the person skilled in the art and do not need to be described in detail here. Furthermore, TTV protein specific antibodies are already available.

[0030] In a preferred embodiment, the methods described herein include the further step of standardizing the amount of viral nucleic acid or protein that is measured. In a preferred embodiment, it may be advantageous to standardize the TTV level, i.e. the amount of TTV DNA, TTV RNA or TTV protein present in a biological sample, to a parameter specific for this sample. The measured TTV amount can be standardized to a certain parameter to reduce the error rate when comparing the viral load of two different biological samples. An example of a parameter that can be useful for this standardization can be a physical parameter that is independent of the content of the sample, such as the volume of the sample. The amount of TTV DNA, TTV RNA or TTV protein can also be standardized to the total amount of DNA, RNA or protein present in the sample. Alternatively, a specific DNA or RNA sequence or a specific protein can be used 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. To this end, the amount of TTV nucleic acid or protein measured in a biological sample can be correlated with a defined amount of a suitable identifiable and quantifiable nucleic acid or protein, such as a host or exogenous nucleic acid or protein. Preferably, this identifiable and quantifiable nucleic acid or protein is treated (e.g., amplified, sequenced, etc.) in the same manner as the target nucleic acid or protein. Thus, from the start, a known amount of this identifiable and quantifiable nucleic acid or protein can be added to the sample, which is then treated like the target nucleic acid or protein and goes through all the sample preparation steps before the amount of said viral nucleic acid or protein is measured. The preparation steps may include measures to protect the viral nucleic acid and destroy the host nucleic acid, for example using different nucleases. Alternatively, these steps may include measures to protect viral proteins and destroy host proteins, for example, using different proteases. The internal control allows the quality and extent of processing (e.g., amplification or sequencing) of the molecule of interest (nucleic acid or protein) in the sample to be evaluated. Preferably, the internal control is a nucleic acid molecule with a known sequence, which is present in the sample at a known concentration. More preferably, the nucleic acid molecule is a circular single-stranded genomic DNA molecule of a virus of known sequence and concentration in the sample. Such a known virus may be, for example, a virus of the Circoviridae family. The absolute number of TTV genomes of known sequence and concentration can be estimated from the ratio of the number of sequences in the sample to the control. Alternatively, the internal control is a protein of known sequence, which is present in the sample at a known concentration.

[0031] Once the amount of TTV has been determined by measuring the amount of TTV nucleic acid or protein determined (the latter optionally being standardized), it may be advantageous to compare it with a predetermined reference TTV amount. In this preferred embodiment, the present disclosure provides a method for assessing the risk of complications in a patient admitted to a medical facility and not undergoing immunosuppressive treatment, in vitro or ex vivo, comprising: a) determining the viral load of at least one Torque Teno Virus (TTV) in a biological sample from said patient; b) comparing the viral load determined for the biological sample with a predetermined reference value; and c) establishing the risk of complications if the viral load determined for the biological sample is greater or less than the predetermined reference value; Includes. For the purposes of this application, "reference TTV amount" or "reference viral load" means any TTV amount used as a reference. This means that the reference TTV amount corresponds to a "reference level of TTV nucleic acid (or protein)" or a "control level of TTV nucleic acid (or protein)", i.e. the concentration of TTV nucleic acid (or protein) used as a standard. "Reference concentration of TTV nucleic acid (or protein)" is intended herein to mean a reference level that can be compared with a test sample and that is measured in a control sample obtained from a subject or group of subjects that exhibits a particular trauma and / or infection condition. It can be, for example, a subject or group of subjects that is healthy or does not exhibit a particular trauma and / or infection. It can also be a patient or group of patients admitted to a medical facility, in particular an intensive care unit, in particular a patient or group of patients that have suffered an immunoinflammatory attack, for example a patient or group of patients that have sepsis, have suffered a burn(s), a trauma, and / or have undergone surgery. Finally, it can be the same individual that has undergone surgery, for example before or immediately after said surgery. The reference level can be determined by several methods. For example, the control can be a pre-defined value that can be in various forms. It can be a single threshold value, such as a median or mean value. The "reference level" can be a single value that is individually applicable to each patient. Alternatively, the reference level can vary based on a particular subpopulation of patients. Thus, for example, the TTV load of an older man may be at a different reference level than that of a younger man, and the viral load of a woman may be at a different reference level than that of a male.Furthermore, the "reference level" may be established based on a comparison group, such as a group without a high level of TTV nucleic acid (or protein) and a group with a high level of TTV nucleic acid (or protein).Another example of a comparison group is a group suffering from a particular disease, condition or symptom and a group without the disease.The predetermined value may be defined, for example, when the test population is divided evenly (or unevenly) 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 levels of TTV nucleic acid (or protein) in a population of patients suffering from a disease or undergoing a treatment that results in an immunosuppressive state. This can be achieved, for example, by analysis by histograms, where the entire cohort of subjects is presented in the form of a graph, with the first axis representing the level of the TTV nucleic acid (or protein) and the second axis representing the number of patients in the patient group that express the TTV nucleic acid (or protein) at a given level. Two or more separate 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 reference level can then be determined based on the level that best distinguishes these separate groups. The reference level can also represent the levels of two or more TTV nucleic acids (or proteins) present. Two or more markers can be represented, for example, by the ratio of the values ​​of the levels of each marker.

[0032] Similarly, a population that is believed to be in good health will have a "normal" range that is different from the range of a population known to be in a condition associated with high concentrations of the TTV nucleic acid (or protein). Thus, the predetermined value selected can take into account the category to which the individual belongs. Appropriate ranges and categories can be easily selected by those skilled in the art through routine experimentation. "High" and "increased" mean higher than the selected control. Generally, the control is based on normal individuals who appear to be in good health and within the appropriate age range. In a preferred embodiment, the reference concentration corresponds to the concentration of a TTV nucleic acid (or protein) or a combination of TTV nucleic acids (or proteins) in the general population. It will also be understood that the control in the methods described herein can be a biological sample that is measured in parallel with the sample being tested, apart from the predetermined value. In this embodiment, the reference level is the level of TTV nucleic acid (or protein) in a sample from a subject in good health. Preferably, the reference concentration of a TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or protein) in a subject in good health or in a population of subjects in good health. In another preferred embodiment, the reference concentration of a TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or protein) in a patient suffering from an immunoinflammatory attack or in a patient group suffering from an immunoinflammatory attack. In another preferred embodiment, the reference concentration of a TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or protein) in a patient suffering from sepsis or in a patient population suffering from sepsis. In another preferred embodiment, the reference concentration of a TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or protein) in a patient suffering from a burn or in a patient population suffering from a burn. In another preferred embodiment, the reference concentration of a TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or protein) in a patient suffering from trauma or in a patient population suffering from trauma. In another preferred embodiment, the reference concentration of a TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or protein) in a patient who has undergone surgery or in a group of patients who have undergone surgery. In another preferred embodiment, the reference concentration of a TTV nucleic acid (or protein) is the concentration of this TTV nucleic acid (or protein) in the same individual who underwent surgery at a particular time point, for example before or immediately after the surgery in question. By comparing the TTV viral load in a biological sample from the patient with a predefined reference value, the patient's risk of complications can be established.

[0033] In a particular embodiment, the predefined reference value corresponds to a viral load in a biological sample from a patient, such as blood or a derivative thereof, such as plasma and / or serum, of more than 7000 copies per ml of biological sample, such as more than 10000 copies, more than 20000 copies, or more than 40000 copies per ml of biological sample. In this particular embodiment, a patient's TTV viral load greater than the predefined reference value correlates with the severity of the patient's condition and may also correlate with an increased risk of developing complications, in particular healthcare associated infections. According to a variant of this particular embodiment, the predetermined reference value corresponds to the TTV viral load of 10000 copies per ml of biological sample.Therefore, according to this variant, when the TTV viral load determined in the biological sample from patient is greater than said predetermined reference value, it is established that there is an increased risk of complications.On the contrary, when the TTV viral load determined in the biological sample from patient is less than said predetermined reference value, it is established that there is no increased risk of complications. A greater amount of TTV in a patient compared to the amount of TTV in an individual or population in good health may be an indication that the patient is at increased risk of a complication.

[0034] The term "greater" as used herein, in certain embodiments, refers to a larger amount, e.g., an amount slightly greater than an original or reference amount, or, e.g., an amount significantly greater than an original or reference amount, particularly any amount within a range. As a variant, "increase" can be 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% greater than the amount or activity to which the increased amount or activity is compared.

[0035] The term "less than" as used herein, in certain embodiments, refers to a smaller amount, e.g., an amount slightly less than an original or reference amount, or, e.g., an amount much less than an original or reference amount, particularly any amount within a range. As a variant, "reducing" can 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 amount or activity to which the reduced amount or activity is compared. Advantageously, as reference TTV viral load the viral load of at least one TTV in the same patient at a second time point may be used.

[0036] In a preferred embodiment, the present disclosure provides a method for in vitro or ex vivo assessment of risk of complications in patients admitted to a medical facility and not undergoing immunosuppressive treatment, comprising: a) measuring a first viral load of at least one TTV in a biological sample of said patient derived from a first sample taken at time T1; b) measuring a second viral load of at least one TTV in a biological sample of said patient derived from a second sample taken at said time T2; c) calculating the variation between the viral load at T2 and the viral load at T1 to obtain a ΔTTV value; and d) to establish conclusions regarding the risk of complications from the results of the comparison; Includes. Surprisingly, the inventors have determined that a variation in the TTV viral load in a patient during their stay in a healthcare facility, i.e. an increase or decrease in the TTV viral load according to the values ​​defined below, correlates with an increased risk of complications for said patient, in particular an increased risk of developing a healthcare associated infection. This method is therefore particularly useful for assessing and monitoring the risk of complications over time in patients admitted to a medical facility. In a preferred embodiment, the first sample in step a) is taken at a time T1 selected 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, where it is understood that the 1st day corresponds to the day after admission and / or the day after the patient suffered an immunoinflammatory attack. In a particular embodiment, when the patient is a patient admitted to a medical facility for surgery, more particularly for major surgery, the first sample in step a) may be taken at the time of surgery, i.e. immediately before, immediately after and / or during the surgical procedure. In a preferred embodiment, the second sample of step b) is taken at a time T2, which second time is later than the first time T1 of step a). Preferably, the second sample is taken between days 8 and 31, more preferably between days 14 and 31. Thus, time T2 is preferably selected from days 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 and 31. In other embodiments, the second sample is taken 31 days after admission of the patient to a medical facility, e.g., 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.

[0037] In one embodiment, the TTV viral load is also measured for a number of samples taken at different times between times T1 and T2. In particular, the TTV viral load is also measured for at least one sample, preferably at least two samples, preferably at least three samples, preferably at least four samples, preferably at least five samples, such as six, seven or eight samples, at different times between times T1 and T2. In one embodiment, the TTV viral load at time T1 corresponds to the average or median viral load measured in multiple biological samples from the patient taken between the day of admission and / or the day of the immunoinflammatory attack and day 7. Preferably, the TTV viral load at time T1 corresponds to the average or median viral load measured in at least two, preferably at least three, preferably at least four biological samples from the patient taken between the day of admission and / or the day of the immunoinflammatory attack and day 7. As a non-limiting example, the TTV viral load at time T1 may correspond to the average or median viral load measured in biological samples from the patient taken on days 1 or 2, 3 or 4, and 5, 6 or 7. In one embodiment, the TTV viral load at time T2 corresponds to the average or median viral load measured in multiple biological samples from the patient taken between 8 and 31 days after hospitalization and / or after the immunoinflammatory challenge. Preferably, the TTV viral load at time T2 corresponds to the average or median viral load measured in at least two, preferably at least three, preferably at least four biological samples from the patient taken between 8 and 31 days after hospitalization and / or after the immunoinflammatory challenge. As a non-limiting example, the TTV viral load at time T2 may correspond to the average or median viral load measured in biological samples from the patient taken on day 14, or any day between days 13 and 18, and on day 28, or any day between days 26 and 36.

[0038] Advantageously, in step c) the ΔTTV value is the variation between the TTV viral load at time T2 and the viral load at time T1: ΔTTV = (TTV viral load at time T2) - (TTV viral load at time T1) Corresponds to. Based on the TTV viral load at time T1 and the TTV viral load at time T2, one skilled in the art can calculate the slope of the line defined by these two measurements. Thus, ΔTTV corresponds to the value of the slope between times T1 and T2. According to the present specification, the TTV viral load between times T1 and T2 is preferably considered stable when the ΔTTV value is within the range of -1.25 to +1.25, preferably within the range of -1.20 to +1.20, preferably within the range of -1.15 to +1.15, preferably within the range of -1.10 to +1.10. For example, the stability of the viral load may correspond to a slope within the range of -1.25 to +1.25 measured between the median viral loads for multiple samples at T1 and T2. Furthermore, the TTV viral load between times T1 and T2 preferably has a ΔTTV value greater than +1.25, preferably greater than +1.35, preferably greater than +1.40, preferably greater than +1.45, more preferably greater than +1.50. Furthermore, the TTV viral load between times T1 and T2 is preferably considered to be decreased if: the TTV value is less than -1.25, preferably less than -1.35, preferably less than -1.40, preferably less than -1.45, more preferably less than -1.50. Thus, if the variation in TTV viral load (DTTV) between times T1 and T2 is within the range of -1.25 to +1.25, in step d) it can be concluded that the patient is not at increased risk for the development of a healthcare-associated infection. On the other hand, if the variation in TTV viral load (DTTV) between times T1 and T2 is not within the range of -1.25 to +1.25, in step d) it can be concluded that the patient is at high risk for the development of a healthcare associated infection.

[0039] Thus, in a preferred embodiment, the method according to the present invention comprises the steps of: a) measuring a first viral load of at least one TTV in a biological sample of said patient derived from a first sample taken at time T1; b) measuring a second viral load of at least one TTV in a biological sample of said patient derived from a second sample taken at said time T2; c) calculating the variation between the viral load at T2 and the viral load at T1 to obtain a ΔTTV value; and d) concluding that there is no increased risk of complications if the DTTV is within the range of -1.25 to +1.25, preferably -1.20 to +1.20, and more preferably -1.10 to +1.10; This includes performing the following:

[0040] In a particularly advantageous embodiment, the present description relates to a method for assessing, in vitro or ex vivo, the risk of complications in a patient admitted to a medical facility and not receiving immunosuppressive treatment, comprising measuring the viral load of at least one Torque Teno Virus (TTV) in a biological sample of said patient, and further detecting the presence of at least one herpesvirus in said biological sample of said patient.

[0041] Indeed, it is to the inventors' credit that they have determined that the simultaneous detection of high viral loads of TTV and at least one HPV in a patient's biological sample, particularly during the first week after admission to an intensive care unit, indicates that said patient has an increased risk of complications, in particular an increased risk of developing a healthcare associated infection. What is particularly surprising is the association between the presence of early TTV / HPV viremia (as early as the first week after admission) and an increased risk of developing a HAI during the first month.

[0042] As used herein, the term "detecting the presence of at least one herpes virus" or "detecting at least one herpes virus" includes qualitative and / or quantitative detection. In various embodiments, HPV is detected by detecting the level of HPV nucleic acid, more preferably HPV DNA. According to the present specification, a herpesvirus is determined to be present or detected if at least one protein from at least one HPV strain, more preferably at least one antigen from at least one HPV strain, is detected in at least one biological sample from the patient. Preferably, a herpesvirus is determined to be present or detected if at least one nucleic acid from at least one HPV strain, more preferably at least one DNA from at least one HPV strain, is detected in at least one biological sample from the patient, referred to as positive viremia or positive DNAemia.

[0043] As used herein, "viremia" refers to the content of viral particles, or viral load, in a biological sample for a given virus. Viremia is positive when the number of detected viral particles is greater than a predetermined threshold (LOD) in the biological sample for a given virus. Conversely, viremia is negative when the viral particles are not detected in the biological sample.

[0044] "DNAemia" means the detection of viral DNA in a biological sample, in particular in a sample of white blood cells isolated from plasma, whole blood and / or peripheral blood and / or in a buffy coat sample (the fraction of a blood sample that does not clotted after centrifugation, containing most of the white blood cells and platelets). Certain techniques are available to detect DNAemia, in particular techniques based on PCR, hybrid capture and analysis of branched DNA. DNAemia is positive if DNA from the virus being tested is detected in the biological sample. Conversely, DNAemia is negative if the viral DNA is not detected in the biological sample. "Viremic event" means that a given virus was detected at least once during the test, in particular that a positive DNAemia for a given virus was detected in at least one biological sample from a patient during the test. In this preferred embodiment, a patient not undergoing immunosuppressive treatment is at increased risk of complications if the TTV viral load in a biological sample from said patient, e.g. blood or a derivative thereof such as plasma and / or serum, is greater than 7000 copies per ml of biological sample, preferably greater than 10000 copies per ml of biological sample, and if the presence of herpes virus is also detected.

[0045] In this preferred embodiment, the method comprises the steps of: a) determining the viral load of at least one TTV in a biological sample of said patient; b) comparing the viral load determined for said biological sample with a predetermined reference value of 10,000 copies / ml; c) detecting the presence of at least one herpes virus, preferably by measuring DNAemia, and d) establishing the presence of an increased risk of complications if the viral load determined for said biological sample is greater than said predetermined reference value and if at least one herpes virus is present; The method may include performing

[0046] In another preferred embodiment, the present specification relates to a method for in vitro or ex vivo assessment of the risk of complications in a patient admitted to a medical facility and not receiving immunosuppressive treatment, comprising measuring the viral load of at least one Torque Teno Virus (TTV) in a biological sample of the patient, and further detecting the presence of at least one herpesvirus in the biological sample of the patient. In the context of the present invention, the "viral load of at least one herpesvirus (HPV)" or "load of at least one HPV" corresponds to the viral load of at least one HPV strain, i.e. the number of viral particles of at least one HPV strain present in a biological sample. The amount of at least one HPV in a patient means the viral load of at least one HPV strain in said patient. The HPV load can be determined by measuring the amount of components, such as nucleic acid or protein, of at least one HPV strain in said biological sample. Preferably, the HPV load corresponds to the amount of nucleic acid sequence of a given HPV strain present in the biological sample. Thus, according to the invention, it is possible to distinguish between different specific HPV strains measured in the biological sample. Preferably, the determination of the HPV load comprises the determination of the amount of active and / or inactive viral copies. It consists in determining the amount of circulating, integrated or latent viral copies. Methods for detecting and / or measuring the viral load of HPV nucleic acid in a biological sample are the same as those previously described for TTV and include, inter alia, amplification, preferably by PCR, more preferably by real-time PCR, sequencing, hybridization with a labeled probe, and all other methods known to the skilled artisan.

[0047] Herpes viruses (Herpesviridae or HPV) are well known to those skilled in the art. They are a family of DNA viruses that affect a variety of animal species, including birds, fish, reptiles, amphibians, and mammals, including humans. All members of the HPV family share a common structure: a relatively large, monosegmented, double-stranded, linear DNA encoding 100-200 genes enclosed within an icosahedral protein cage called the capsid, which is itself surrounded by a protein layer called the coat, which contains both viral proteins and viral mRNA, and a lipid bilayer membrane called the envelope. All HPVs are nuclear replicative, meaning that the viral DNA is transcribed into mRNA in the nucleus of the infected cell, where it can remain indefinitely in a latent form. Nine types of HPV are known to primarily infect humans. At least five are highly prevalent in humans: herpes simplex 1 and 2 (HSV-1 and HSV-2, also known as HHV-1 and HHV-2), which together can cause labial and genital herpes; varicella zoster (or HHV-3), which causes chickenpox and shingles; Epstein-Barr virus (EBV or HHV-4), which is responsible for certain diseases including mononucleosis and some cancers; and human cytomegalovirus (HCMV or CMV or HHV-5), which is highly prevalent in humans. More than 90% of adults are infected with at least one of these viruses, and the virus remains latent in almost all infected people. 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). As used herein, "herpes virus" or "HPV" or "HHV" (human herpes virus) refers to a virus of the Herpesviridae family. As used herein, "HPV genome" refers to the genome of all Herpesviridae families, including those of types HSV-1, HSV-2, HHV-3, EBV, CMV, HHV-6, HHV-7 and KSHV, more specifically those of types CMV, EBV, HHV6 and HSV-1. In a preferred embodiment, the 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 herein to the genomes of herpesvirus strains of the types CMV AD169, EBV B-95, HSV1 95 and HHV6 Z29. As a non-limiting example, the genome of CMV AD169 may be that whose sequence is referenced under Genbank Accession No. X17403.1. Similarly, the genome of EBV B-95 may be that whose sequence is referenced under Genbank Accession No. V01555.2. An exemplary genome of. HHV6 Z29 may be that whose sequence is referenced under Genbank Accession No. X83413.2.

[0048] Preferentially, the presence of at least one HPV is determined and / or the HPV viral load is measured using real-time quantitative PCR. Numerous methods for detecting and quantifying HPV have been described in the art (for example, Walton et al., Plos ONE 9(6):e98819, 2014). In particular, reference is made to the methods described in the following documents: More specifically, in Table S1 of Non-Patent Document 4, reproduced here in Figures 6 and 7. This method is particularly advantageous for its simplicity and robustness; it can also be carried out using any type of PCR platform. It is based on the amplification of genes specific for each of these herpesviruses, namely ppUL83, which codes for a structural protein of CMV, the gene BXLF1, which codes for a thymidine kinase of EBV, the gene US7, which codes for a glycoprotein of HSV-1, and the gene U57, which codes for a capsid protein of HHV6. To carry out this method, it is particularly advantageous to use R-GENE® diagnostic kits (CMV R-GENE® Reference No. 69-003B, EBV R-GENE® Reference No. 69-002B, HSV1 HSV2 VZV R-GENE® Reference No. 69-004B and HVV6 R-GENE® Reference No. 69-100B, bioMerieux, France). In a preferred embodiment, the method described herein further comprises the step of normalizing the amount of HPV nucleic acid measured.To this end, the skilled artisan can refer to the methods and embodiments described above for normalizing TTV levels.These methods can be easily adapted to normalize the amount of HPV nucleic acid. When the amount of at least one HPV is determined by measuring the amount of HPV nucleic acid (the latter is optionally standardized), it may be advantageous to compare it with a reference HPV amount.For this purpose, those skilled in the art can refer to the above-mentioned methods and embodiments regarding comparison with TTV reference level.These methods can be easily adapted to comparison with reference HPV level.

[0049] Methods for Treating Patients Suspected of Being at Risk for Complications Thus, a patient's susceptibility to complications, particularly the risk of infection, can be determined using the methods described herein, allowing treatments to be developed and tailored specifically to the patient's needs. Thus, advantageously, the method, in all its embodiments, may comprise managing medical care, in particular to reduce the risk of complications, in particular the risk of developing a healthcare associated infection. Patients identified as being at high risk for developing a healthcare-associated infection may receive appropriate managed care with the goal of reducing the risk of developing the healthcare-associated infection, e.g., to reduce the risk of developing sepsis, septic shock, or the risk of death. Thus, in another aspect, the present invention also provides a method for treating a patient suspected of being at risk for a complication, comprising: - identifying patients at risk for complications by implementing any one of the above embodiments; and - Adapting the management of the patient's health care identified in the preceding paragraph to reduce the risk of deterioration. Examples of managing health care can include immunomodulatory therapy tailored to the patient, or prophylactic antibiotic therapy, a combination of both therapies, and / or directed to a high dependency unit or resuscitation department the day after measuring the expression of the biomarker, to reduce the risk of developing a health care associated infection, for example to reduce the risk of developing sepsis, septic shock, or even death. Preferably, the immunomodulatory treatment is an immunostimulatory therapy if the individual is determined to be immunosuppressed, or an anti-inflammatory therapy if the individual is determined to be inflammatory. Among the immunostimulatory treatments that may be selected, mention may be made, by way of example, of the interleukins, in particular IL-7, IL-15 or IL-3, growth factors, in particular GM-CSF, interferons, in particular IFN-γ, anti-PD 1 antibodies, anti-PDL 1 antibodies, anti-LAG 3 antibodies, anti-TIM 3 antibodies, anti-IL-10 antibodies or anti-CTLA 4 antibodies), transferrin and apoptosis inhibitor molecules, FLT3L, thymosin alpha 1, and adrenergic antagonists. Among the anti-inflammatory treatments, glucocorticoids, cytostatics, molecules acting on immunophilins and cytokines, molecules blocking the IL-1 receptor, and anti-TNF treatments can be mentioned in particular. Examples of suitable prophylactic antibiotic treatments to prevent pneumonia are described in particular in Annales Francaises d'Anesthesie et de Reanimation (30; 2011; 168-190). In other words, rather than being in a department with close monitoring that is not necessary for the patient, the patient can be sent quickly to an outpatient department, for example an infectious diseases department, without the risk of developing a healthcare-associated infection. The invention will be more precisely described by the following examples. EXAMPLES

[0050] To better understand the physiopathology underlying the viral reactivation phenomenon in patients admitted to intensive care units, a study was performed in a cohort of patients involved in the REALISM clinical trial (NCT01931956). In each of these patients, blood DNAemia of the TTV anellovirus and the herpesviruses CMV, EBV, HHV6 and HSV-1 was measured over a period of one month after admission to intensive care. The expected results were aimed at identifying common complications associated with the viral reactivation phenomenon in these patients and to be able to identify whether one or more viruses may be associated with the occurrence of a particular complication.

[0051] Materials and Methods Study population The observational study was performed on a prospective cohort of critically ill patients presenting with sepsis, severe trauma, severe burns, or undergoing planned surgery at the Anesthesia and Intensive Medicine Department of the Édouard Herriot Hospital (Hospits Cials de Lyon, France). The inclusion period was 28 months (December 2015–March 2018). The study protocol was approved by the Institutional Ethics Committee (Comite de Protection des Personnes Sud-Est II) under number 2015-42-2. The clinical trial was also registered at clinicaltrials.gov (NCT02638779). At the time of inclusion, written informed consent was obtained from each healthy volunteer and each patient. In cases where the patient was unable to consent directly, informed consent was obtained from the patient's legal representative and reconfirmed with the patient at the earliest possible opportunity. Inclusion criteria were as follows: patients aged 18 years or older, clinical diagnosis of sepsis as defined by the 2016 SEPSIS-3 consensus definition (Singer et al., 2016), severe trauma with an Injury Severity Score (ISS) >15, severe burns with a total burn surface area of ​​>30%, or surgical patients undergoing major surgery such as esophagogastroctomy, Brier cystectomy, head pancreaticoduodenectomy and / or abdominal aortic aneurysm surgery via laparotomy. Exclusion criteria were: the presence of pre-existing disorders or procedures that may affect the patient's immune status; in particular, patients under immunosuppressive treatment, pregnant patients, institutionalized patients and patients who were unable to give informed consent were excluded. In parallel, a cohort of 175 healthy volunteers (81 men and 94 women) aged between 18 and 82 years was prospectively recruited. To take into account possible age- and sex-related influences on immune parameters, the distribution of healthy volunteers was based on demographic data on age and sex of the French population in 2016.

[0052] Sampling and Data Collection For each patient, samples and clinical data were collected three times during the first week after admission: (1) days 1 or 2 (D1–2), (2) days 3 or 4 (D3–4) and (3) D5, D6 or D7 (D5–7), then two more times during the month after admission: (4) D14 and (5) D28. For healthy volunteers, samples were taken and clinical data were recorded during the study visits. Data collection was described elsewhere (Venet et al., 2021). Patient demographic data, comorbidities, diagnoses, severity and clinical outcomes were recorded prospectively manually. Longitudinal monitoring was performed for 30 days. In each case, whole peripheral blood from each patient and each healthy volunteer was processed within 3 hours after the sample was drawn. Blood was collected into EDTA tubes for measurement of plasma viral DNAemia, immunophenotyping by flow cytometry and plasma cytokine levels, into two heparin tubes for functional tests (proliferation, cytokine production experiments) and finally into blood RNA PAXgene tubes (PreAnalytix, Hilden, Germany) for measurement of biomarker mRNA concentrations in whole blood by real-time PCR using EvaGreen or TaqMan probe methodologies. PAXgene samples were stabilized at ambient temperature for at least 2 hours after sampling and then frozen at -80°C according to the manufacturer's recommendations.

[0053] Definition of Complications The main complications evaluated in the context of this study were secondary infections (healthcare associated infections - HAI) at day 28 (D28) and mortality at D28. Other complications evaluated included total length of stay in the intensive care unit (ICU) and hospital stay, daily use of invasive medical devices (endotracheal intubation, indwelling urinary catheters and central venous lines). Information collected regarding infections was reviewed by an independent decision-making committee of three clinicians not involved in the recruitment or care of patients in the study. Confirmation of the diagnosis of secondary infections (healthcare-associated infections, HAIs) by this committee was based on the guidelines of the European Society of Clinical Microbiology and Infectious Diseases and the Infectious Diseases Society of America. Only the first episode of secondary infection was considered for the analysis.

[0054] Determination of viral DNAemia The semi-automated process for determining viral DNAemia of TTV and the four herpesviruses integrates standardized procedures for sample processing and real-time quantitative PCR and has been described in detail previously (Non-Patent Document 4). Briefly, viral DNA was extracted from plasma samples using the Maxwell® HT Viral TNA chemistry kit (Promega) composed of paramagnetic silica particles and the Freedom EVO® liquid handling robot (TECAN) according to the manufacturer's instructions. PCR controls were added to each plasma sample before extraction. The limit of detection (LOD) of the semi-automated process was previously determined to be the minimum amount that could be distinguished from the absence of detection (blank value) using a viral standard (Non-Patent Document 4). The detection limit of TTV was determined using plasma samples containing a given amount of TTV. First, the control plasma sample was serially diluted from 10-1 to 10-4, and each diluted sample was extracted independently three times, and then amplified using quantitative PCR to determine the approximate detection limit. Second, the highest dilution that gave a positive signal was diluted again to 1 / 2 and 1 / 4, and then each diluted sample was extracted independently 20 times and amplified. The most diluted positive replicate with 100% detection was used as the detection limit. The LODs, expressed as copies per ml of plasma, are 167 for TTV, 100 for CMV, 33 for EBV, 166 for HHV6, and 33 for HSV1. Viral DNA was amplified using real-time PCR on a StepOnePlus™ Real-Time PCR System (ThermoFisher SCIENTIFIC) with R-GENE® Assay Kits using TaqMan probes for CMV, EBV, HSV1, HHV6, and TTV (bioMerieux SA). All nucleic acid samples, randomly distributed on the plate, were amplified simultaneously with quantitative standards, sensitivity controls and negative controls according to the manufacturer's instructions. Finally, the standard curve was used to convert the Ct ("cycle threshold") values ​​for each sample into copies / μl of viral DNA in the PCR reaction tubes, which were then converted into copies / ml of viral DNA in the plasma samples.

[0055] statistical analysis Viremia was measured by measuring DNAemia for each virus, and a viremic event, i.e. positive DNAemia, was characterized by a copy number of virus per microliter greater than a predefined threshold (greater than LOD). Positive DNAemia was considered early if positive DNAemia was detected for at least one sample from a patient during the first week (days 1 / 2, 3 / 4 or 5 / 7) after the patient was admitted to intensive care. DNAemia was also evaluated on D14 and D28. Data are presented in the form of numbers and percentages (qualitative variables) and medians and 25 / 75th percentiles (quantitative variables).

[0056] Associations between viraemic events and clinical complications (supra (Non-Patent Document 4), survival gained in intensive care or bacterial infections) were assessed using chi-square tests or Fisher's exact tests, where appropriate (Non-Patent Document 4). Binary associations between DNAemia / viremia events and quantitative immune markers (cells, cytokines, mRNA) were analyzed using Wilcoxon signed-rank tests. Finally, analyses were performed using R software, version 3.4.4, with statistical significance defined as an alpha risk of incorrectly rejecting the null hypothesis of 5% (p<0.05 indicates a significant association).

[0057] result Cohort description The REALISM cohort of 377 critically ill patients consisted of 107 cases of sepsis, 137 cases of severe trauma, 24 cases of severe burns, and 109 cases of major surgery. Quantitative data (viral load) rather than qualitative data (DNAemia) may better distinguish between (1) insignificant viral load, (2) viral “reactivation” as a putative marker of immunosuppression, and (3) high viral load (<1%) supporting the presence of actual viral infection requiring treatment (Textoris et al., 2017). Patients with high TTV DNAemia (viral load >10,000 copies / ml) had more severe disease, higher severity scores, and greater need for treatment (Figure 3). An increase in TTV viral load (p=0.025) was significantly correlated with a longer stay in intensive care, whereas both increases (p=0.048) and decreases (p=0.048) in TTV viral load, i.e., fluctuations in TTV viral load, were significantly associated with the occurrence of healthcare-associated infections acquired in intensive care during the first month (Figures 2A and 2B).

[0058] Moreover, we surprisingly found that cases of HAIs tended to be more common in patients with TTV and positive DNAemia for at least one herpesvirus than in patients with positive herpes DNAemia only (Figures 2A and 2B).

[0059] DNAemia in intensive care unit patients during the first week and first month of hospitalization Week 1 TTV was detected (positive DNAemia) in 217 (58%) of the patients in the cohort, i.e. slightly more than in healthy controls (51%). TTV was detected alone in 160 (42%) patients and was simultaneously detected in patients with one (n=48, 13%), two (n=6, 2%), or three or more herpes viruses (n=3, 1%). The most common herpesvirus detected concomitantly with TTV 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 (whether herpesvirus or TTV) (n=189) was similar to the number of patients with positive DNAemia for the presence of at least two viruses (n=188).

[0060] First month TTV was detected in 228 (60%) patients in the cohort. 151 (40%) patients had positive DNAemia for TTV only, whereas TTV was simultaneously detected (positive DNAemia) in patients with one (n=59, 16%), two (n=11, 3%) or three or more (n=7, 2%) herpesviruses. All herpesviruses were simultaneously detected with TTV, with EBV (n=36, 10%) being the most common co-detection, followed by HSV1 (n=28, 7%), CMV (n=23, 6%) and HHV6 (n=19, 5%). Overall, the cohort contained an equal number of patients with monoviral infections (positive DNAemia) (n=187) as those with concomitant infections (n=190).

[0061] The proportion of patients with TTV was similar in patients with sepsis, severe trauma, and major surgery (56–64%) and slightly lower in burn victims (46%), compared with 51% observed in healthy volunteers. As a trend, the proportion of patients with high TTV titers appears to be greater in the surgical patient population, and severity criteria correlate with high TTV viral loads, particularly >7000 copies / ml (Figure 4).

[0062] Association of DNAemia with clinical complications At the end of the 28-day observation period, the occurrence of at least one episode of secondary healthcare-associated infection will be considered. The individual detection of each herpesvirus during the first week of hospitalization, unlike during the first month, is not significantly associated with the risk of healthcare-associated infection (HAI). In the absence of positive DNAemia for TTV, a significant increase in the rate of HAI is observed in patients who had at least one viraemic episode (positive DNAemia) during the first month for any herpesvirus considered individually. Furthermore, positive DNAemia for TTV alone (without detection of herpesvirus) is associated with a reduced incidence of HAI (Figure 3). We also observed that a stable TTV viral load during the first month was associated with a reduced incidence of HAIs ( Fig. 5 ). When considering the viral DNAemia of patients from the cohort in the first month, a higher proportion of HAI is observed in patients who had a viremic event (positive DNAemia for two or more herpesviruses or for TTV and at least one herpesvirus) than in patients who had positive DNAemia for a single virus (22%) or no detected DNAemia (19%) (chi-square test, p<0.001). When considering events of positive viral DNAemia in week 1, patients with a viremic event had a similar HAI rate (11%) as patients with positive DNAemia of a single virus (9%) or without any detectable DNAemia (9%) (chi-square test, P = 0.8). Surprisingly, the results show that the concomitant detection of high TTV viral load (especially >10 000 copies per ml of sample) and positive DNAemia for herpesviruses early, as early as the first week after patient admission, is significantly associated with the risk of developing a healthcare-associated infection (Figure 3).

[0063] In summary, with regard to viremia, disease group and admission criteria, (i) the proportion of patients with fluctuating (increasing or decreasing) TTV viral loads over time tend to be higher in patients with sepsis than in other groups, and (ii) positive DNAemia for herpes as well as high TTV viral loads appear to be associated with the severity of the patient's condition on admission. With regard to HAI, (i) the simultaneous detection of a high TTV viral load and positive DNAemia for herpes, preferably as early as the first week of hospitalization, correlates with the risk of more frequent occurrence of HAI, and (ii) a stable TTV viral load is associated with a relatively low occurrence of HAI, unlike fluctuations in TTV viral load, which also correlate with the risk of occurrence of HAI in patients.

[0064] Consideration The results demonstrate that insignificant TTV amounts can be distinguished from high TTV amounts. It is therefore particularly to the inventors' credit that a TTV expression threshold associated with physiopathological features in intensive care patients has been identified. In summary, the results presented above demonstrated that (i) detection in patients of high TTV loads associated with herpesvirus reactivation as early as the first week of admission to intensive care was associated with an increased risk of HAI, and (ii) early and high TTV viremia was associated with immune system impairment.

[0065] Changes in viremia over time were less discernible for TTV (from 58% in the first week to 60% over one month), and positive viremia for TTV was more frequent for TTV alone than for simultaneous detection of TTV and herpesvirus (42% vs. 15% measured during the first week and 40% vs. 20% measured during the first month). The prevalence of TTV is higher in men in cohorts of individuals with the condition (Focosi et al., 2020) and transplant recipients (Bal et al., 2018), and no gender bias was observed in the study population, although there was a male predominance in the subgroup with TTV viral loads >10,000 copies / ml. TTV and EBV were the most frequently detected viruses simultaneously, 8%-10% during the first week and first month, respectively.

[0066] During the same period, only the variation in TTV load was significantly associated with an increased risk of HAI occurrence. Indeed, the mere presence of TTV, even at high loads, is insufficient to determine an increased risk of HAI. Nevertheless, patients with multiple viremic events, including those with positive TTV DNAemia during the first month after hospitalization, have a higher frequency of HAI (44%) than those with a single or no viremic event (22% and 19%, respectively). Thus, quite surprisingly and unexpectedly, high TTV load and simultaneous detection of herpesviruses in the first week of hospitalization are significantly associated with the occurrence of HAI events during the first month. In conclusion, the results demonstrate that a correlation exists between viral reactivation, especially TTV, in combination with or without HPV, and the risk of developing a healthcare-associated infection in patients admitted to intensive care units.

[0067] References

[0068] [Table 1]

Claims

1. A method for in vitro or ex vivo assessment of the risk of complications in a patient admitted to a medical facility and not undergoing immunosuppressive therapy, comprising the following: a) measuring the viral load of at least one torque teno virus (TTv) in a biological sample of said patient; b) comparing the viral load determined for said biological sample with a predetermined reference value; and c) establishing the risk of complications when the viral load determined for said biological sample is greater than or less than said predetermined reference value. A method comprising the above.

2. The method according to claim 1, wherein said predetermined reference value is 10,000 copies / ml.

3. The method according to claim 2, wherein when the viral load determined for said biological sample is greater than said predetermined reference value, the presence of an increased risk of complications is established.

4. The method according to claim 1 or 2, further comprising detecting the presence of at least one herpes virus by measuring DNAemia.

5. The method according to claim 4, wherein when the viral load of at least one TTV determined for said biological sample is more than 10,000 copies / ml and at least one herpes virus is present, the presence of an increased risk of complications is established.

6. a) measuring a first viral load of at least one TTV in a biological sample of said patient derived from a first sample taken at time T1; b) measuring a second viral load of at least one TTV in a biological sample of said patient derived from a second sample taken at time T2; c) calculating the variation between the viral load at T2 and the viral load at T1 to obtain a ΔTTV value; and d) establishing a conclusion regarding the risk of complications from the result of the comparison. A method comprising the above, according to claim 1.

7. The method according to claim 6, wherein when the ΔTTV value is within the range of -1.25 to +1.25, -1.20 to +1.20, or -1.10 to +1.10, a conclusion is drawn that the risk of complications does not increase.

8. The method according to claim 1, wherein the risk of complications is the risk of healthcare-associated infection (HAI) occurring.

9. The method according to claim 1, wherein said biological sample is derived from a patient admitted to a hospital, emergency department, intensive care unit, intensive care unit or high-dependency facility.

10. The method according to claim 1, wherein the biological sample is derived from a patient with septic condition, a patient with septic shock, a patient with burns, a patient with severe burns, a patient with trauma, a patient with severe trauma, a patient who has undergone surgery, or a patient who has undergone major surgery.

11. The method according to claim 1, wherein the biological sample is derived from a patient with septic condition, a patient with burns, a patient with trauma, a patient who has undergone surgery, and a patient admitted to the intensive care unit.

12. The method according to claim 4, wherein at least one herpes virus is selected from the group consisting of CMV, EBV, HHV6 and HSV-1, or is EBV.

13. The method according to claim 1, wherein the biological sample is a body fluid derived from a patient, which is selected from the group consisting of blood or its derivatives, plasma and / or serum, cerebrospinal fluid, urine, and bronchoalveolar lavage fluid.

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