Viral DNAemia and DNA Virus Seropositivity and Mortality in Pediatric Sepsis.

Importance: Sepsis is a leading cause of pediatric mortality. Little attention has been paid to the association between viral DNA and mortality in children and adolescents with sepsis. Objective: To assess the association of the presence of viral DNA with sepsis-related mortality in a large multicenter study. Design, Setting, and Participants: This cohort study compares pediatric patients with and without plasma cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 (HSV-1), human herpesvirus 6 (HHV-6), parvovirus B19 (B19V), BK polyomavirus (BKPyV), human adenovirus (HAdV), and torque teno virus (TTV) DNAemia detected by quantitative real-time polymerase chain reaction or plasma IgG antibodies to CMV, EBV, HSV-1, or HHV-6. A total of 401 patients younger than 18 years with severe sepsis were enrolled from 9 pediatric intensive care units (PICUs) in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Collaborative Pediatric Critical Care Research Network. Data were collected from 2015 to 2018. Samples were assayed from 2019 to 2022. Data were analyzed from 2022 to 2023. Main Outcomes and Measures: Death while in the PICU. Results: Among the 401 patients included in the analysis, the median age was 6 (IQR, 1-12) years, and 222 (55.4%) were male. One hundred fifty-four patients (38.4%) were previously healthy, 108 (26.9%) were immunocompromised, and 225 (56.1%) had documented infection(s) at enrollment. Forty-four patients (11.0%) died in the PICU. Viral DNAemia with at least 1 virus (excluding TTV) was detected in 191 patients (47.6%) overall, 63 of 108 patients (58.3%) who were immunocompromised, and 128 of 293 (43.7%) who were not immunocompromised at sepsis onset. After adjustment for age, Pediatric Risk of Mortality score, previously healthy status, and immunocompromised status at sepsis onset, CMV (adjusted odds ratio [AOR], 3.01 [95% CI, 1.36-6.45]; P = .007), HAdV (AOR, 3.50 [95% CI, 1.46-8.09]; P = .006), BKPyV (AOR. 3.02 [95% CI, 1.17-7.34]; P = .02), and HHV-6 (AOR, 2.62 [95% CI, 1.31-5.20]; P = .007) DNAemia were each associated with increased mortality. Two or more viruses were detected in 78 patients (19.5%), with mortality among 12 of 32 (37.5%) who were immunocompromised and 9 of 46 (19.6%) who were not immunocompromised at sepsis onset. Herpesvirus seropositivity was common (HSV-1, 82 of 246 [33.3%]; CMV, 107 of 254 [42.1%]; EBV, 152 of 251 [60.6%]; HHV-6, 253 if 257 [98.4%]). After additional adjustment for receipt of blood products in the PICU, EBV seropositivity was associated with increased mortality (AOR, 6.10 [95% CI, 1.00-118.61]; P = .049). Conclusions and Relevance: The findings of this cohort study suggest that DNAemia for CMV, HAdV, BKPyV, and HHV-6 and EBV seropositivity were independently associated with increased sepsis mortality. Further investigation of the underlying biology of these viral DNA infections in children with sepsis is warranted to determine whether they only reflect mortality risk or contribute to mortality.

Distinct Immune Reconstitution Profiles Captured by Immune Functional Assays at 6 Months Post Allogeneic Hematopoietic Stem Cell Transplantation.

Immune reconstitution after allogeneic-hematopoietic-stem-cell transplantation (allo-HSCT) is a complex and individual process. In this cross-sectional study, whole-blood (WB) immune functional assay (IFA) was used to characterize immune function by assessing immune-related gene/pathway alterations. The usefulness of this tool in the context of infection, 6 months after transplantation, was evaluated. Sixty allo-HSCT recipients at 6 months after transplantation and 10 healthy volunteers (HV) were included. WB was stimulated in standardized TruCulture tubes using lipopolysaccharides and Staphylococcal enterotoxin B. Gene expression was quantified using a custom 144-gene panel using NanoString nCounter technology and analyzed using Ingenuity Pathway Analysis. The relationships between immune function and clinical characteristics, immune cell counts, and post-transplantation infections were assessed. Allo-HSCT recipients were able to activate similar networks of the innate and adaptive immune response compared to HV, with, nevertheless, a lower intensity. A reduced number and a lower expression of genes associated with immunoregulatory and inflammatory processes were observed in allo-HSCT recipients. The use of immunosuppressive treatments was associated with a protracted immune reconstitution revealed by transcriptomic immunoprofiling. No difference in immune cell counts was observed among patients receiving or not receiving immunosuppressive treatments using a large immunophenotyping panel. Moreover, the expression of a set of genes, including CCL3/CCL4, was significantly lower in patients with Herpesviridae reactivation (32%, 19/60), which once again was not identified using classical immune cell counts. Transcriptional IFA revealed the heterogeneity among allo-HSCT recipients with a reduced immune function, a result that could not be captured by circulating immune cell counts. This highlights the potential added value of this tool for the personalized care of immunocompromised patients.

Prospective multicentre study of host response signatures in neonatal sepsis in Sub Saharan Africa.

Few biomarkers for sepsis diagnosis are commonly used in neonatal sepsis. While the role of host response is increasingly recognized in sepsis pathogenesis and prognosis, there is a need for evaluating new biomarkers targeting host response in regions where sepsis burden is high and medico-economic resources are scarce. The objective of the study is to evaluate diagnostic and prognostic accuracy of biomarkers of neonatal sepsis in Sub Saharan Africa. This prospective multicentre study included newborn infants delivered in the Abomey-Calavi region in South Benin and their follow-up from birth to 3 months of age. Accuracy of transcriptional (CD74, CX3CR1), proteic (PCT, IL-6, IL-10, IP-10) biomarkers and clinical characteristics to diagnose and prognose neonatal sepsis were measured. At delivery, cord blood from all consecutive newborns were sampled and analysed, and infants were followed for a 12 weeks' period. Five hundred and eighty-one newborns were enrolled. One hundred and seventy-two newborns developed neonatal sepsis (29.6%) and death occurred in forty-nine infants (8.4%). Although PCT, IL-6 and IP-10 levels were independently associated with sepsis diagnosis, diagnostic accuracy of clinical variables combinations was similar to combinations with biomarkers and superior to biomarkers alone. Nonetheless, CD74, being the only biomarkers independently associated with mortality, showed elevated prognosis accuracy (AUC > 0.9) either alone or in combination with other biomarkers (eg. CD74/IP-10) or clinical criterion (eg. Apgar 1, birth weight). These results suggest that cord blood PCT had a low accuracy for diagnosing early onset neonatal sepsis in Sub Saharan African neonates, while association of clinical criterion showed to be more accurate than any biomarkers taken independently. At birth, CD74, either associated with IP-10 or clinical criterion, had the best accuracy in prognosing sepsis mortality.Trial registration Clinicaltrial.gov registration number: NCT03780712. Registered 19 December 2018. Retrospectively registered.

Performance Evaluation of Host Biomarker Combinations for the Diagnosis of Serious Bacterial Infection in Young Febrile Children: A Double-Blind, Multicentre, Observational Study.

The diagnosis of serious bacterial infection (SBI) in young febrile children remains challenging. This prospective, multicentre, observational study aimed to identify new protein marker combinations that can differentiate a bacterial infection from a viral infection in 983 children, aged 7 days-36 months, presenting with a suspected SBI at three French paediatric emergency departments. The blood levels of seven protein markers (CRP, PCT, IL-6, NGAL, MxA, TRAIL, IP-10) were measured at enrolment. The patients received the standard of care, blinded to the biomarker results. An independent adjudication committee assigned a bacterial vs. viral infection diagnosis based on clinical data, blinded to the biomarker results. Computational modelling was applied to the blood levels of the biomarkers using independent training and validation cohorts. Model performances (area under the curve (AUC), positive and negative likelihood ratios (LR+ and LR-)) were calculated and compared to those of the routine biomarkers CRP and PCT. The targeted performance for added value over CRP or PCT was LR+ ≥ 5.67 and LR- ≤ 0.5. Out of 652 analysed patients, several marker combinations outperformed CRP and PCT, although none achieved the targeted performance criteria in the 7 days-36 months population. The models seemed to perform better in younger (7-91 day-old) patients, with the CRP/MxA/TRAIL combination performing best (AUC 0.895, LR+ 10.46, LR- 0.16). Although computational modelling using combinations of bacterial- and viral-induced host-protein markers is promising, further optimisation is necessary to improve SBI diagnosis in young febrile children.

A Simple-to-Perform ifn-γ mRNA Gene Expression Assay on Whole Blood Accurately Appraises Varicella Zoster Virus-Specific Cell-Mediated Immunity After Allogeneic Hematopoietic Stem Cell Transplantation.

Herpes zoster, which is due to the reactivation of Varicella zoster virus (VZV), is a leading cause of morbidity after allogeneic hematopoietic stem cell transplantation (allo-HSCT). While cell-mediated immunity (CMI) is critical to inhibiting VZV reactivation, CMI is not routinely assessed due to a lack of reliable tests. In this study, we aimed to evaluate VZV-specific CMI among allo-HSCT recipients (n = 60) and healthy individuals (HI, n = 17) through a panel of three immune functional assays after ex vivo stimulation by VZV antigen: quantification of (i) IFN-γ release in the supernatants, (ii) T-cell proliferation after a 7-day stimulation of peripheral blood mononuclear cells (PBMC), and (iii) measurement of the ifn-γ mRNA gene expression level after 24 h of stimulation of a whole-blood sample. VZV responsiveness was defined according to IFN-γ release from VZV-stimulated PBMC. Upon VZV stimulation, we found that allo-HSCT recipients at a median time of 6 [5-8] months post-transplant had lower IFN-γ release (median [IQR], 0.34 [0.12-8.56] vs. 409.5 [143.9-910.2] pg/ml, P <.0001) and fewer proliferating T cells (0.05 [0.01-0.57] % vs. 8.74 [3.12-15.05] %, P <.0001) than HI. A subset of allo-HSCT recipients (VZV-responders, n = 15/57, 26%) distinguished themselves from VZV-non-responders (n = 42/57, 74%; missing data, n = 3) by higher IFN-γ release (80.45 [54.3-312.8] vs. 0.22 [0.12-0.42] pg/ml, P <.0001) and T-cell proliferation (2.22 [1.18-7.56] % vs. 0.002 [0.001-0.11] %, P <.0001), suggesting recovery of VZV-specific CMI. Interestingly, VZV responders had a significant fold increase in ifn-γ gene expression, whereas ifn-γ mRNA was not detected in whole blood of VZV-non-responders (P <.0001). This study is the first to suggest that measurement of ifn-γ gene expression in 24-h-stimulated whole blood could be an accurate test of VZV-specific CMI. The routine use of this immune functional assay to guide antiviral prophylaxis at an individual level remains to be evaluated.

A stratification strategy to predict secondary infection in critical illness-induced immune dysfunction: the REALIST score.

BACKGROUND: Although multiple individual immune parameters have been demonstrated to predict the occurrence of secondary infection after critical illness, significant questions remain with regards to the selection, timing and clinical utility of such immune monitoring tests. RESEARCH QUESTION: As a sub-study of the REALISM study, the REALIST score was developed as a pragmatic approach to help clinicians better identify and stratify patients at high risk for secondary infection, using a simple set of relatively available and technically robust biomarkers. STUDY DESIGN AND METHODS: This is a sub-study of a single-centre prospective cohort study of immune profiling in critically ill adults admitted after severe trauma, major surgery or sepsis/septic shock. For the REALIST score, five immune parameters were pre-emptively selected based on their clinical applicability and technical robustness. Predictive power of different parameters and combinations of parameters was assessed. The main outcome of interest was the occurrence of secondary infection within 30 days. RESULTS: After excluding statistically redundant and poorly predictive parameters, three parameters remained in the REALIST score: mHLA-DR, percentage of immature (CD10- CD16-) neutrophils and serum IL-10 level. In the cohort of interest (n = 189), incidence of secondary infection at day 30 increased from 8% for patients with REALIST score of 0 to 46% in patients with a score of 3 abnormal parameters, measured ad D5-7. When adjusted for a priori identified clinical risk factors for secondary infection (SOFA score and invasive mechanical ventilation at D5-7), a higher REALIST score was independently associated with increased risk of secondary infection (42 events (22.2%), adjusted HR 3.22 (1.09-9.50), p = 0.034) and mortality (10 events (5.3%), p = 0.001). INTERPRETATION: We derived and presented the REALIST score, a simple and pragmatic stratification strategy which provides clinicians with a clear assessment of the immune status of their patients. This new tool could help optimize care of these individuals and could contribute in designing future trials of immune stimulation strategies.

Evaluation of acute kidney injury by urinary tissue inhibitor metalloproteinases-2 and insulin-like growth factor-binding protein 7 after pediatric cardiac surgery.

BACKGROUND: With adult patients, the measurement of [TIMP-2]*[IGFBP7] can predict the risk of moderate to severe AKI within 12 h of testing. In pediatrics, however, the performance of [TIMP-2]*[IGFBP7] as a predictor of AKI was less studied and yet to be widely utilized in clinical practice. This study was conducted to validate the utility of [TIMP-2]*[IGFBP7] as an earlier biomarker for AKI prediction in Chinese infants and small children. METHODS: We measured urinary [TIMP-2]*[IGFBP7] using NEPHROCHECK® at eight perioperative time points in 230 patients undergoing complex cardiac surgery and evaluated the performance of [TIMP-2]*[IGFBP7] for predicting severe AKI within 72 h of surgery. RESULTS: A total of 50 (22%) of 230 developed AKI stages 2-3 within 72 h after CPB initiation. In the AKI stage 2-3 patients, two patterns of serum creatinine (SCr) elevations were observed. The patients with only a transient increase in SCr within 24 h (< 24 h, early AKI 2-3) did not experience a worse outcome than patients in AKI stage 0-1. AKI stage 2-3 patients with SCr elevation after 24 h (24-72 h, late AKI 2-3), as well as AKI dialysis patients (together designated severe AKI), did experience worse outcomes. Compared to AKI stages 0-1, significant elevations of [TIMP-2]*[IGFBP7] values were observed in severe AKI patients at hours T2, T4, T12, and T24 following CPB initiation. The AUC for predicting severe AKI with [TIMP-2]*[IGFBP7] at T2 (AUC = 0.76) and maximum T2/T24 (AUC = 0.80) are higher than other time points. The addition of the NEPHROCHECK® test to the postoperative parameters improved the risk assessment of severe AKI. CONCLUSIONS: Multiple AKI phenotypes (early versus late AKI) were identified after pediatric complex cardiac surgery according to SCr-based AKI definition. Urinary [TIMP-2]*[IGFBP7] predicts late severe AKI (but not early AKI) as early as 2 h following CPB initiation. A higher resolution version of the Graphical abstract is available as Supplementary information.

Herpes DNAemia and TTV Viraemia in Intensive Care Unit Critically Ill Patients: A Single-Centre Prospective Longitudinal Study.

Introduction: We analysed blood DNAemia of TTV and four herpesviruses (CMV, EBV, HHV6, and HSV-1) in the REAnimation Low Immune Status Marker (REALISM) cohort of critically ill patients who had presented with either sepsis, burns, severe trauma, or major surgery. The aim was to identify common features related to virus and injury-associated pathologies and specific features linking one or several viruses to a particular pathological context. Methods: Overall and individual viral DNAemia were measured over a month using quantitative PCR assays from the 377 patients in the REALISM cohort. These patients were characterised by clinical outcomes [severity scores, mortality, Intensive Care Unit (ICU)-acquired infection (IAI)] and 48 parameters defining their host response after injury (cell populations, immune functional assays, and biomarkers). Association between viraemic event and clinical outcomes or immune markers was assessed using χ2-test or exact Fisher's test for qualitative variables and Wilcoxon test for continuous variables. Results: The cumulative incidence of viral DNAemia increased from below 4% at ICU admission to 35% for each herpesvirus during the first month. EBV, HSV1, HHV6, and CMV were detected in 18%, 12%, 10%, and 9% of patients, respectively. The incidence of high TTV viraemia (>10,000 copies/ml) increased from 11% to 15% during the same period. Herpesvirus viraemia was associated with severity at admission; CMV and HHV6 viraemia correlated with mortality during the first week and over the month. The presence of individual herpesvirus during the first month was significantly associated (p < 0.001) with the occurrence of IAI, whilst herpesvirus DNAemia coupled with high TTV viraemia during the very first week was associated with IAI. Herpesvirus viraemia was associated with a lasting exacerbated host immune response, with concurrent profound immune suppression and hyper inflammation, and delayed return to immune homeostasis. The percentage of patients presenting with herpesvirus DNAemia was significantly higher in sepsis than in all other groups. Primary infection in the hospital and high IL10 levels might favour EBV and CMV reactivation. Conclusion: In this cohort of ICU patients, phenotypic differences were observed between TTV and herpesviruses DNAemia. The higher prevalence of herpesvirus DNAemia in sepsis hints at further studies that may enable a better in vivo understanding of host determinants of herpesvirus viral reactivation. Furthermore, our data suggest that EBV and TTV may be useful as additional markers to predict clinical deterioration in ICU patients.

Evaluation of TTV replication as a biomarker of immune checkpoint inhibitors efficacy in melanoma patients.

Torque Teno Virus (TTV) is a small, non-enveloped, single-stranded and circular DNA virus that infects the majority of the population worldwide. Increased levels of plasma TTV viral load have been observed in various situations of immune deficiency or dysregulation, and several studies have suggested that TTV levels may be inversely correlated with immune competence. The measurement of TTV viremia by qPCR has been proposed as a potential biomarker for the follow-up of functional immune competence in immunosuppressed individuals, particularly hematopoietic stem cell transplant recipients. We hypothesized that TTV viral load could be used as a prognostic marker of immune checkpoint inhibitor (ICI) efficacy, and therefore investigated the TTV viral load in melanoma patients treated with nivolumab or pembrolizumab before and after 6 months of treatment. In the present study, TTV viral load was not different in melanoma patients before anti-PD-1 introduction compared to healthy volunteers, was not modified by ICI treatment and did not allowed to distinguish patients with treatment-sensitive tumor from patients with treatment-resistant tumor.

Differential response induced by LPS and MPLA in immunocompetent and septic individuals.

Lipopolysaccharide (LPS) and monophosphoryl lipid A (MPLA) induce, overall, similar transcriptional profiles in healthy individuals, although LPS has been shown to more potently induce pro-inflammatory cytokines. We explore herein whether MPLA could be considered as a synthetic replacement of LPS in immune functional assays to study anergy of immune cells in septic patients. Ex vivo whole blood stimulation with MPLA revealed a lower induction of the TNFα secreted protein in 20 septic patients (SP) compared to 10 healthy volunteers (HV), in agreement with monocyte anergy. Principal component analysis of the 93-gene molecular response to MPLA and LPS stimulation found that the main variability was driven by stimulation in HV and by pathophysiology in SP. MPLA was a stronger inducer of the HLA family genes than LPS in both populations, arguing for divergent signalling pathways downstream of TLR-4. In addition, MPLA appeared to present a more informative stratification potential within the septic population.

Concomitant Assessment of Monocyte HLA-DR Expression and Ex Vivo TNF-α Release as Markers of Adverse Outcome after Various Injuries-Insights from the REALISM Study.

Intensive care unit (ICU) patients develop an altered host immune response after severe injuries. This response may evolve towards a state of persistent immunosuppression that is associated with adverse clinical outcomes. The expression of human leukocyte antigen DR on circulating monocytes (mHLA-DR) and ex vivo release of tumor necrosis factor α (TNF-α) by lipopolysaccharide-stimulated whole blood are two related biomarkers offered to characterize this phenomenon. The purpose of this study was to concomitantly evaluate the association between mHLA-DR and TNF-α release and adverse clinical outcome (i.e., death or secondary infection) after severe trauma, sepsis or surgery in a cohort of 353 ICU patients. mHLA-DR and TNF-α release was similarly and significantly reduced in patients whatever the type of injury. Persistent decreases in both markers at days 5-7 (post-admission) were significantly associated with adverse outcomes. Overall, mHLA-DR (measured by flow cytometry) appears to be a more robust and standardized parameter. Each marker can be used individually as a surrogate of immunosuppression, depending on center facilities. Combining these two parameters could be of interest to identify the most immunosuppressed patients presenting with a high risk of worsening. This last aspect deserves further exploration.

Torque Teno Virus Viral Load as a Marker of Immune Function in Allogeneic Haematopoietic Stem Cell Transplantation Recipients.

Torque teno virus (TTV) has been proposed as a surrogate biomarker of T-cell function in allogeneic-haematopoietic-stem-cell transplantation (allo-HSCT). Conflicting data exists regarding the value of TTV to assess the degree of immunosuppression. The aim of the present study was to investigate the correlation between TTV viral load and immune function. Using samples from a prospective cohort composed of healthy-volunteers (HV) and allo-HSCT recipients at 6 months post-transplantation, we assessed the correlation between TTV viraemia and immune cell counts or T-cell proliferation capacity post-phytohaemagglutinin stimulation. TTV viraemia was detected in 68% of HV (n = 80) and 100% of allo-HSCT recipients (n = 41; p < 0.001); it was significantly higher in allo-HSCT recipients (3.9 vs. 2.1 Log copies/mL, p < 0.001). There was no correlation between T-cell function and CD3+T-cell count (rho: 0.002) suggesting that T-cell count can normalise without full functional recovery. Furthermore, no significant correlation was observed between TTV viraemia and absolute total/subset lymphocyte counts (rho: <0.13). The highest correlation was observed between TTV viral load and T-cell proliferation capacity (rho: -0.39). We therefore report an inverse correlation between T-cell function and TTV viraemia that is independent of T-cell count. Monitoring of TTV viraemia could be a fast suitable option to objectively assess the competence of immune function in at-risk populations.

Deciphering heterogeneity of septic shock patients using immune functional assays: a proof of concept study.

The complexity of sepsis pathophysiology hinders patient management and therapeutic decisions. In this proof-of-concept study we characterised the underlying host immune response alterations using a standardised immune functional assay (IFA) in order to stratify a sepsis population. In septic shock patients, ex vivo LPS and SEB stimulations modulated, respectively, 5.3% (1/19) and 57.1% (12/21) of the pathways modulated in healthy volunteers (HV), highlighting deeper alterations induced by LPS than by SEB. SEB-based clustering, identified 3 severity-based groups of septic patients significantly different regarding mHLA-DR expression and TNFα level post-LPS, as well as 28-day mortality, and nosocomial infections. Combining the results from two independent cohorts gathering 20 HV and 60 patients, 1 cluster grouped all HV with 12% of patients. The second cluster grouped 42% of patients and contained all non-survivors. The third cluster grouped 46% of patients, including 78% of those with nosocomial infections. The molecular features of these clusters indicated a distinctive contribution of previously described genes defining a "healthy-immune response" and a "sepsis-related host response". The third cluster was characterised by potential immune recovery that underlines the possible added value of SEB-based IFA to capture the sepsis immune response and contribute to personalised management.

Towards standardization of immune functional assays.

Recent advances in the immunotherapy field require evaluation of the immune function to adapt therapeutic decisions. Immune functional assays (IFA) are able to reveal the immune status and would be useful to further adapt and/or improve patient's care. However, standardized methods are needed to implement IFA in clinical settings. We carried out an independent validation of a published method used to characterize the underlying host response to infectious conditions using an IFA. We evaluated the reproducibility and robustness of this IFA and the associated readout using an independent healthy volunteers (HV) cohort. Expression of a 44-gene signature and IFNγ protein secretion was assessed after stimulation. We observed a strong host-response correlation between the two cohorts. We also highlight that standardized methods for immune function evaluation exist and could be implemented in larger-scale studies. This IFA could be a relevant tool to reveal innate and adaptive immune dysfunction in immune-related disorders patients.

Metagenomic Investigation of Torque Teno Mini Virus-SH in Hematological Patients.

A new member of Anelloviridae, named torque teno mini virus (TTMV)-SH, was recently identified in the serum of three Hodgkin's lymphoma patients suggesting that TTMV-SH may be associated with this type of hematological malignancy. We investigated by metagenomic analysis the presence of TTMV-SH-related viruses in plasma samples (n = 323) collected from patients with various hematological malignancies (multiple myeloma (MM, n = 256), non-Hodgkin's lymphoma (NHL, n = 20), acute myeloid leukemia (n = 10)) and from healthy donors (n = 37). TTMV-SH-related strains were identified in 24 samples corresponding to four MM and one NHL patients. Phylogenic analysis revealed that the 24 isolates were close to the TTMV-SH strains previously identified, sharing 79.6-86.7% ORF1 nucleotide sequence identity. These results suggest that TTMV-SH-related viruses might be found in hematological diseases other than Hodgkin's lymphoma. Due to the high genetic variability within Anelloviridae species, the association between a particular medical condition and a new genotype should be interpreted with caution.

VaccHemInf project: protocol for a prospective cohort study of efficacy, safety and characterisation of immune functional response to vaccinations in haematopoietic stem cell transplant recipients.

INTRODUCTION: Immune reconstitution after haematopoietic stem cell transplantation (HSCT) is a complex and dynamic process, varying from a state of nearly complete immunosuppression to an expected full immune recovery. Specific vaccination guidelines recommend reimmunisation after HSCT but data regarding vaccine efficacy in this unique population are scarce. New immune functional assays could enable prediction of vaccine response in the setting of HSCT. METHODS AND ANALYSIS: A prospective, longitudinal single-centre cohort study of autologous and allogeneic HSCT recipients was designed in order to determine the vaccine response to five vaccine targets (pneumococcus, hepatitis B virus, Haemophilus Influenzae type b, tetanus and diphtheria) and to correlate it to immune function parameters. A workflow was set up to study serological response to vaccines and to describe the functional immune status of 100 HSCT recipients (50 autologous and 50 allogeneic) before and 3, 12 and 24 months after primary immunisation. At each time point, 'basic' immune status recording (serology, immunophenotyping of lymphocyte subsets by flow cytometry) will be assessed. The immune response will furthermore be evaluated before and 3 months after primary vaccination by two ex vivo immune functional assays assessing: (1) tumour necrosis factor alpha, interferon gamma production and host messenger RNA expression on whole-blood stimulation by lipopolysaccharide or Staphylococcus aureus enterotoxin B and (2) T-lymphocyte proliferation in response to a standard mitogen (phytohaemagglutinin) or to selected recall antigens. Reference intervals will be determined from a cohort of 30 healthy volunteers. This translational study will provide data describing vaccine response, immune functionality of HSCT recipients over time and will allow mapping HSCT recipients with regard to their immune function. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the institutional review board (no 69HCL17_0769). Results will be communicated at scientific meetings and submitted for publication in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT03659773; Pre-results.

Metagenomic Next-Generation Sequencing Reveals Individual Composition and Dynamics of Anelloviruses during Autologous Stem Cell Transplant Recipient Management.

Over recent years, there has been increasing interest in the use of the anelloviruses, the major component of the human virome, for the prediction of post-transplant complications such as severe infections. Due to an important diversity, the comprehensive characterization of this viral family over time has been poorly studied. To overcome this challenge, we used a metagenomic next-generation sequencing (mNGS) approach with the aim of determining the individual anellovirus profile of autologous stem cell transplant (ASCT) patients. We conducted a prospective pilot study on a homogeneous patient cohort regarding the chemotherapy regimens that included 10 ASCT recipients. A validated viral mNGS workflow was used on 108 plasma samples collected at 11 time points from diagnosis to 90 days post-transplantation. A complex interindividual variability in terms of abundance and composition was noticed. In particular, a strong sex effect was found and confirmed using quantitative PCR targeting torque teno virus, the most abundant anellovirus. Interestingly, an important turnover in the anellovirus composition was observed during the course of the disease revealing a strong intra-individual variability. Although more studies are needed to better understand anellovirus dynamics, these findings are of prime importance for their future use as biomarkers of immune competence.

LTR-retrotransposon transcriptome modulation in response to endotoxin-induced stress in PBMCs.

BACKGROUND: Human Endogenous Retroviruses (HERVs) and Mammalian apparent LTR-retrotransposons (MaLRs) represent the 8% of our genome and are distributed among our 46 chromosomes. These LTR-retrotransposons are thought to be essentially silent except in cancer, autoimmunity and placental development. Their Long Terminal Repeats (LTRs) constitute putative promoter or polyA regulatory sequences. In this study, we used a recently described high-density microarray which can be used to study HERV/MaLR transcriptome including 353,994 HERV/MaLR loci and 1559 immunity-related genes. RESULTS: We described, for the first time, the HERV transcriptome in peripheral blood mononuclear cells (PBMCs) using a cellular model mimicking inflammatory response and monocyte anergy observed after septic shock. About 5.6% of the HERV/MaLR repertoire is transcribed in PBMCs. Roughly one-tenth [5.7-13.1%] of LTRs exhibit a putative constitutive promoter or polyA function while one-quarter [19.5-27.6%] may shift from silent to active. Evidence was given that some HERVs/MaLRs and genes may share similar regulation control under lipopolysaccharide (LPS) stimulation conditions. Stimulus-dependent response confirms that HERV expression is tightly regulated in PBMCs. Altogether, these observations make it possible to integrate 62 HERVs/MaLRs and 26 genes in 11 canonical pathways and suggest a link between HERV expression and immune response. The transcriptional modulation of HERVs located close to genes such as OAS2/3 and IFI44/IFI44L or at a great distance from genes was discussed. CONCLUSION: This microarray-based approach revealed the expression of about 47,466 distinct HERV loci and identified 951 putative promoter LTRs and 744 putative polyA LTRs in PBMCs. HERV/MaLR expression was shown to be tightly modulated under several stimuli including high-dose and low-dose LPS and Interferon-γ (IFN-γ). HERV incorporation at the crossroads of immune response pathways paves the way for further functional studies and analyses of the HERV transcriptome in altered immune responses in vivo such as in sepsis.

Detection of single nucleotide polymorphisms by minisequencing on a polypyrrole DNA chip designed for medical diagnosis.

With the increasing availability of genetic information and its relationship to human diseases, there is a growing need in the medical diagnostic field for technologies that can proceed to the parallel genotyping of multiple markers. In this paper, we report the development of a new flexible microarray-based method that aims to be inexpensive, accurate, and adapted to routine analysis. The construction of the MICAM (MICrosystem for Analysis in Medicine) DNA chip is based on the controlled electro-synthesis of a conducting polymer film bearing oligonucleotide probes on gold electrodes. First, accessible 3'OH-ends of grafted probes are directly used to conduct single template-dependent nucleotide extension reactions with fluorescence-labeled chain terminators. Then, the fluorescence of incorporated dideoxynucleotides on controls and probes of interest are recorded to assess base calling. Here, we present the development of the methodology to assign the genotype of TP53 (tumor protein p53) codon 72 polymorphism and its application to analysis of genomic DNA from cell lines and from human colorectal samples. The genotyping results obtained by mini-sequencing on the polypyrrole DNA chip were 100% concordant with data obtained by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Moreover, the developed probe array assay has been successfully applied to the detection of TP53 loss of heterozygosity.