Search

Background Critical illness such as sepsis is a life-threatening syndrome defined as a dysregulated host response to infection and is characterized by patients exhibiting impaired immune response. In the field of diagnosis, a gap still remains in identifying the immune profile of critically ill patients in the intensive care unit (ICU). Methods A new multiplex immune profiling panel (IPP) prototype was assessed for its ability to semiquantify messenger RNA immune-related markers directly from blood, using the FilmArray System, in less than an hour. Samples from 30 healthy volunteers were used for the technical assessment of the IPP tool. Then the tool was clinically assessed using samples from 10 healthy volunteers and 20 septic shock patients stratified using human leukocyte antigen–DR expression on monocytes (mHLA-DR). Results The IPP prototype consists of 16 biomarkers that target the immune response. The majority of the assays had a linear expression with different RNA inputs and a coefficient of determination (R2) > 0.8. Results from the IPP pouch were comparable to standard quantitative polymerase chain reaction and the assays were within the limits of agreement in Bland–Altman analysis. Quantification cycle values of the target genes were normalized against reference genes and confirmed to account for the different cell count and technical variability. The clinical assessment of the IPP markers demonstrated various gene modulations that could distinctly differentiate 3 profiles: healthy volunteers, intermediate mHLA-DR septic shock patients, and low mHLA-DR septic shock patients. Conclusions The use of IPP showed great potential for the development of a fully automated, rapid, and easy-to-use immune profiling tool. The IPP tool may be used in the future to stratify critically ill patients in the ICU according to their immune status. Such stratification will enable personalized management of patients and guide treatments to avoid secondary infections and lower mortality.

Background: Few biomarkers for sepsis diagnosis are commonly used in neonatal sepsis. Whilst host response is increasingly recognized in sepsis pathogenesis and prognosis, there is a need for evaluating the accuracy of 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. Methods: Prospective multicentre study conducted between April 2016 and March 2018 in three University hospitals and two sub-urbans healthcare centres in the south Benin region. Patients were followed 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. Results: From April 17, 2016 through March 12, 2018, a total of 581 newborns were enrolled. One hundred and seventy-two newborns developed neonatal sepsis (29.6%) and death occurred in forty-nine infants (8.4%). Among all tested biomarkers and clinical criteria, CD74/IP-10 ratio showed the best accuracy for neonatal sepsis diagnosis, while PCT accuracy was low. Among biomarkers and clinical criteria studied, only CD74 and PCT were independently associated with mortality, with CD74 showing an elevated predictive accuracy. Conclusion: Cord blood PCT had a low accuracy for diagnosing early onset neonatal sepsis in Sub Saharan African neonates, while CD74/IP-10 ratio had the best diagnostic accuracy. CD74 expression at birth had the best accuracy in prognosing sepsis mortality.Trial registration: Clinicaltrial.gov registration number: NCT03780712. Registered 19 December 2018. Retrospectively registered

Influenza A viruses are amongst the most challenging viruses that threaten both human and animal health. Constantly evolving and crossing species barrier, the emergence of novel zoonotic pathogens is one of the greatest challenges to global health security. During the last decade, considerable attention has been paid to influenza virus infections in dogs, as two canine H3N8 and H3N2 subtypes caused several outbreaks through the United States and Southern Asia, becoming endemic. Cats, even though less documented in the literature, still appear to be susceptible to many avian influenza infections. While influenza epidemics pose a threat to canine and feline health, the risks to humans are largely unknown. Here, we review most recent knowledge of the epidemiology of influenza A viruses in dogs and cats, existing evidences for the abilities of these species to host, sustain intraspecific transmission, and generate novel flu A lineages through genomic reassortment. Such enhanced understanding suggests a need to reinforce surveillance of the role played by companion animals-human interface, in light of the “One Health” concept and the potential emergence of novel zoonotic viruses.

BACKGROUND According to the Developmental Origins of Health and Diseases concept, exposures in the preconception period may be critical. For the first time, we evaluated the effect of preconception poor anthropometric status on infant's growth in sub-Saharan Africa. METHODS A mother-child cohort was followed prospectively from preconception to 1 year old in Benin. Maternal anthropometric status was assessed by prepregnancy body mass index (BMI), approximated by BMI at the first antenatal visit before 7 weeks' gestation, and gestational weight gain (GWG). BMI was categorized as underweight, normal, overweight, and obesity according to World Health Organization standards. GWG was categorized as low ( 12 kg). In infant, stunting and wasting were defined as length-for-age and weight-for-length z scores less than -2 SD, respectively. We evaluated the association between BMI/GWG and infant's weight and length at birth and during the first year of life, as well as with stunting and wasting at 12 months using mixed linear and logistic regression models. RESULTS In multivariate, preconceptional underweight was associated with a lower infant's weight at birth and during the first year (-164 g; 95% CI, -307 to -22; and -342 g; 95% CI, -624 to -61, respectively) and with a higher risk of stunting at 12 months (adjusted odds ratio [aOR] = 3.98; 95% CI, 1.01-15.85). Furthermore, preconceptional obesity and a high GWG were associated with a higher weight and length at birth and during the first year. CONCLUSION Underweight and obesity before conception as well as GWG were associated with infant's growth. These results argue for preventive interventions starting as early as the preconception period to support child long-term health.

BackgroundCritical illness such as sepsis is a life-threatening syndrome defined as a dysregulated host response to infection and is characterized by patients exhibiting various impaired immune profiles. In the field of diagnosis, a gap still remains in identifying the immune profile of critically-ill patients in the ICU. The availability of an immune profiling tool holds a great potential in providing patients at high risk with more accurate and precise management. In this study, a multiplex immune profiling panel prototype was assessed for its ability to semi-quantify immune markers directly from blood, using the FilmArray® System.ResultsThe Immune Profiling Panel (IPP) prototype consists of 16 biomarkers that target both the innate and adaptive immune responses, pro- and anti-inflammatory mediators as well as genes involved in diverse regulatory pathways. The analytical studies carried out on healthy volunteers showed minimal inter- and intra-variability in testing the samples across the tested lots. The majority of the assays were linear with an R2 higher than 0.8. Results from the IPP pouch were comparable to qPCR and were within the limits of agreement. Finally, quantification cycle values of the target genes were normalized against reference genes to account for the different composition of cells among specimens. The use of the selected panel of markers in IPP demonstrated various gene modulations that could distinctly differentiate three profiles: healthy, borderline mHLA-DR septic shock patients and low mHLA-DR septic shock patients.ConclusionThe Immune Profiling Panel allowed host transcriptomic analysis of immune response biomarkers directly from whole blood in less than an hour. The use of IPP showed great potential for the development of a fully automated, rapid and easy-to-use immune profiling tool, enabling the stratification of critically-ill patients at high risk in the ICU.

IntroductionNeonatal sepsis outreaches all causes of neonatal mortality worldwide and remains a major societal burden in low and middle income countries. In addition to limited resources, endemic morbidities, such as malaria and prematurity, predispose neonates and infants to invasive infection by altering neonatal immune response to pathogens. Nevertheless, thoughtful epidemiological, diagnostic and immunological evaluation of neonatal sepsis and the impact of gestational malaria have never been performed.Methods and analysisA prospective longitudinal multicentre follow-up of 580 infants from birth to 3 months of age in urban and suburban Benin will be performed. At delivery, and every other week, all children will be examined and clinically evaluated for occurrence of sepsis. At delivery, cord blood systematic analysis of selected plasma and transcriptomic biomarkers (procalcitonin, interleukin (IL)-6, IL-10, IP10, CD74 and CX3CR1) associated with sepsis pathophysiology will be evaluated in all live births as well as during the follow-up, and when sepsis will be suspected. In addition, whole blood response to selected innate stimuli and extensive peripheral blood mononuclear cells phenotypic characterisation will be performed. Reference intervals specific to sub-Saharan neonates will be determined from this cohort and biomarkers performances for neonatal sepsis diagnosis and prognosis tested.Ethics and disseminationEthical approval has been obtained from the Comité d’Ethique de la Recherche – Institut des Sciences Biomédicales Appliquées (CER-ISBA 85 - 5 April 2016, extended on 3 February 2017). Results will be disseminated through international presentations at scientific meetings and publications in peer-reviewed journals.Trial registration numberClinicalTrials.gov registration number: NCT03780712.

International audience; Integrons are able to recruit resistance genes through integrase-driven recombination events that are regulated by the bacterial SOS response and require the repressor LexA. Class 1 integrons genes are expressed from a common promoter, Pc, of which at least 5 predominant variants, classified from weak to strong, have been described. In Escherichia coli, there is an intertwined regulation between gene cassette expression and integrase activity: the stronger the promoter is, the weaker the integrase is. Class 1 integrons have been frequently described in Acinetobacter baumannii. However, Acinetobacter spp. lack the LexA repressor suggesting that the integrase is constitutively expressed. We characterized the integron content of 83 clinical and environmental A. baumannii strains. We found a predominance of Pc variants described as strong in E. coli. The Pc expression level was 2 to 4-fold lower in A. baumannii than in E. coli, and the diversity of the gene cassette array was low. In A.baumannii integrons with a PcS promoter might have been selected to allow a sufficient resistance level while avoiding the toxicity of a highly active integrase. Furthermore, a transcriptional interference between PcS and PintI1 (as shown in E.coli) may limit the expression of the integrase and thus counterbalance the lack of LexA-driven integrase repression to prevent the cost of the integrase.

Background Ventilator-associated pneumonia (VAP) is one of the most commonly encountered hospital-acquired infections worldwide, and one of the major contributors to an over mortality in critically ill patients. Initial empirical antimicrobial therapy is often broad-spectrum. Fast identification and quantification of microorganisms is of great importance to enable early effective targeted antimicrobial treatment. This trial compares the performance of the new BioFire® Pneumonia Panel (BPP) with quantitative conventional culture (CC) and an independent real-time quantitative molecular-based method (MM), in Intensive Care Unit (ICU) patients with VAP suspicion. Methods Bronchoalveolar lavage (BAL) specimens from 120 patients with suspected VAP, enrolled at four different French ICUs, during January to November 2013, were analysed by CC, following microbiological standard procedures, by BPP and MM. A total of 15 bacterial targets, commonly detected by the three methods, were analysed for concordance above an agreed threshold for positivity. While every step is fully integrated, from specimen-to-results (BPP), bacterial DNA was extracted from each sample on the NucliSENS easyMAG® Platform, and real-time polymerase chain reactions were run in an ABI 7500 Dx thermocycler (MM). Results A total of 117 different BAL specimens were processed. Positive culture was obtained for 65.8% of BAL, while positive detections were observed in 79.4% with BPP and 75.4% with independent MM. Fourteen different species were detected by the three methods, with majority of the bacteria being S. aureus, P. aeruginosa, and H. influenzae. Overall concordance performance between BPP and CC was 89.0% (83.1%–94.9%) positive percentage agreement (PPA) and 95.9% (95.0%–96.9%) negative percentage agreement (NPA). Overall concordance between BPP and MM was 97.1% (93.8%–100.3%) PPA and 96.6% (95.6%–97.6%) NPA. Following discrepancy analyses overall performance increased to 95.3% (91.2–99.3%) PPA when comparing BPP to CC. Conclusion The new BioFire® Pneumonia Panel provides reliable quantitative microbiological data in BAL specimens, in only 65 minutes, which can lead to more appropriate management of VAP suspected patients in the ICU. RUO products used in this study have not been evaluated by the FDA or other regulatory agencies for In Vitro Diagnostic use. Disclosures A. Iannello, bioMérieux: Employee, Salary. C. Dubost, bioMérieux: Employee, Salary. C. Weber, bioMérieux: Employee, Salary. C. Alberti-Segui, bioMérieux: Employee, Salary. C. Mousset, bioMérieux: Employee, Salary. C. Ginocchio, bioMérieux: Employee, Salary. M. Rogatcheva, BioFire: Employee, Salary. V. Moucadel, bioMérieux: Employee, Salary. J. Yugueros-Marcos, bioMérieux: Employee, Salary.

We conducted a retrospective study to evaluate the usefulness of immunoglobulin G (IgG) subclasses against Candida cell wall fragments (CW) and phosphopeptidomannan (PPM) for the diagnosis of invasive candidiasis (IC). We analyzed 54 patients with IC (n = 19), Candida heavy colonization (HC; n = 16), and controls (no IC or HC, n = 19).In nonneutropenic patients (n = 47), the sensitivity and specificity values of IgG1 anti-CW and IgG2 anti-PPM in IC were 88%, 59%, and 88%, 94%, respectively. The areas under the receiver operating characteristic curves were 0.69 (0.51-0.88) and 0.901 (0.78-1.02), respectively. IgG1 mean values (arbitrary units) and 95% confidence interval were 46 (20-71), 42 (-0.38 to 84) and 20 (8.3-32) in IC, HC, and in controls, respectively, and discriminated IC but not HC from controls (P = .032, and P = .77, respectively). IgG2 mean values were 26 (9.2-42), 19 (4.4-33), and 3.2 (0.28-6.6) in IC, HC, and in controls, respectively, and discriminated both IC and HC from controls (P < .0001 and P = .035, respectively) but did not separate IC from HC (P = .2). IgG2 showed positivity as early as one day after the IC diagnosis. Antibodies were detected in only two out of a total of seven neutropenic patients.For both IC and HC patients, the diagnostic performance of IgG2 anti-PPM was better than the one of IgG1 anti-CW. In nonneutropenic patients, IgG2 anti-PPM accurately identified not only IC patients but also HC patients at high risk for IC. This marker may help clinicians in the initiation of early preemptive therapy.

The aroA gene has been identified as a target in screening for the presence of most Aeromonas species so far described by PCR. Synthetic oligonucleotide primers of 24 and 25 nucleotides were used by PCR assay to amplify a sequence of the aroA gene, which encodes 3-phosphoshikimate-1-carboxyvinyltransferase, a key enzyme of aromatic amino acids and folate biosynthetic pathway. A 1236-bp DNA fragment, representing most of the aroA gene, according to the nucleotide sequence of A. salmonicida, was amplified from all Aeromonas species tested, which represented most of the 14 hybridization groups. HaeII digestion of the 1236-bp fragment generated a restriction fragment length polymorphisms which could be used as a powerful tool for identification of aeromonads to the genus level.

Fungi and yeasts are critical causes of acute infection. As such, the detection and identification of these organisms are crucial in the diagnosis of affected patient populations. There is a vast array of commercial tests currently available for diagnostic purposes. These vary from traditional culture and biochemical methods to advanced multiparameter molecular tests. Recent technological advances have driven the development of rapid tests which are complementing and in some cases replacing the more traditional methods of detection. Irrespective of the method used the ultimate goal is timely detection of the infectious agent allowing appropriate treatment and improved outcome for the patient.

Ventilator-associated pneumonia (VAP) is the most common infection in critically ill patients. Initial antibiotic therapy is often broad spectrum, which promotes antibiotic resistance so new techniques are under investigation to obtain early microbiological identification and quantification. This trial compares the performance of a new real-time quantitative molecular-based method with conventional culture in patients with suspected VAP. Patients with suspected VAP who were ventilated for at least 48 h were eligible. An endotracheal aspirate (ETA) and a bronchoalveolar lavage (BAL) were performed at each suspected VAP episode. Both samples were analysed by conventional culture and molecular analysis. For the latter, bacterial DNA was extracted from each sample and real-time PCR were run. In all, 120 patients were finally included; 76% (91) were men; median age was 65 years, and clinical pulmonary infection score was ≥6 for 73.5% (86) of patients. A total of 120 BAL and 103 ETA could be processed and culture results above the agreed threshold were obtained for 75.0% (90/120) of BAL and 60.2% (62/103) of ETA. The main isolated bacteria were Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae. Performance was 89.2% (83.2%-93.6%) sensitivity and 97.1% (96.1%-97.9%) specificity for BAL samples and 71.8% (61.0%-81.0%) sensitivity and 96.6% (95.4%-97.5%) specificity for ETA samples when the molecular biology method was compared with conventional culture method (chosen as reference standard). This new molecular method can provide reliable quantitative microbiological data and is highly specific with good sensitivity for common pathogens involved in VAP.

Fungi and yeasts are critical causes of acute infection. As such, the detection and identification of these organisms are crucial in the diagnosis of affected patient populations. There is a vast array of commercial tests currently available for diagnostic purposes. These vary from traditional culture and biochemical methods to advanced multiparameter molecular tests. Recent technological advances have driven the development of rapid tests which are complementing and in some cases replacing the more traditional methods of detection. Irrespective of the method used the ultimate goal is timely detection of the infectious agent allowing appropriate treatment and improved outcome for the patient.

Background Dengue fever is the most important arthropod born viral disease of public health significance. Although most patients suffer only from flu-like symptoms, a small group of patient experiences more severe forms of the disease. To contribute to a better understanding of its pathogenesis this study aims to identify proteins differentially expressed in a pool of five viremic plasma from severe dengue patients relative to a pool of five non-severe dengue patients. Results The use of Isotope Coded Protein Labeling (ICPLTM) to analyze plasma depleted of twenty high-abundance proteins allowed for the identification of 51 differentially expressed proteins, which were characterized by mass spectrometry. Using quantitative ELISA, three of these proteins (Leucine-rich glycoprotein 1, Vitamin D binding-protein and Ferritin) were confirmed as having an increased expression in a panel of severe dengue plasma. The proteins identified as overexpressed by ICPLTM in severe dengue plasma involve in clear up action after cell injury, tissue coherence and immune defense. Conclusion This ICPLTM study evaluating differences between acute severe dengue plasmas and acute non-severe dengue plasmas suggests that the three proteins identified are overexpressed early in the course of the disease. Their possible use as biomarkers for the prognostic of disease severity is discussed.

Major concern for intubated patients is ventilator-associated pneumonia (VAP). Early detection of VAP and its causative microorganism(s) is a key challenge for clinicians. Diagnosis is based on clinical, radiological, and microbiological elements, the latter being provided 24–48h after sampling. According to practices, clinicians can sample endotracheal aspirates (ETAs) so as to check for patient colonization or perform ETA in case of VAP suspicion. In this proof-of-concept study, we report the evaluation of a semiautomated molecular method to rapidly quantify Staphylococcus aureus , one of the most involved microorganisms in VAP, directly from raw ETA samples. After evaluation using artificial ETA samples, our method was applied on 40 clinical ETA samples. All S . aureus –positive samples were successfully detected and quantified. Our method can provide an efficient sample preparation protocol for all raw ETA samples, combined with an accurate quantification of the bacterial load, in less than 3h 30min.

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 × 10 2 CFU) when S. aureus cells were suspended in sterilized whole milk. Taq I digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.

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. Graphical Abstract

Les infections nosocomiales, ou infections contractées au cours de soins hospitaliers, ont un impact majeur de santé publique. La prévalence de ces infections est de 7,1% dans les pays industrialisés, et de 10% à 15% dans les pays en voie de développement. Les unités de soins intensifs sont les services les plus touchés. Ces infections sont causées principalement par des bactéries. Le développement des infections nosocomiales s'accompagne de morbi-mortalités importantes, entraînant des surcoûts hospitaliers estimés à 7 milliards d'euros par an en Europe, 6,5 milliards de dollars par an aux USA. Ces infections nosocomiales sont pourtant évitables dans plus d'un cas sur cinq. L'augmentation de ces morbi-mortalités est en partie associée à la mise en place d'une antibiothérapie empirique, bien souvent inadaptée et sans bénéfice clinique, à cause du temps nécessaire à l'examen microbiologique. En effet, les méthodes conventionnelles d'identification de pathogènes et de résistances aux antibiotiques, basées sur la culture des pathogènes, sont longues et laborieuses, avec un résultat obtenu plus de 2 à 5 jours après le prélèvement. Des techniques de diagnostic rapide existent, permettant de réduire ce délai à moins de 24h. Parmi ces techniques, sont décrites dans cette thèse la PCR en temps réel, la technique de FISH, la spectrométrie de masse MALDI-TOF et finalement la PCR couplée à la spectrométrie de masse, PCR-ESI. Ces techniques présentent de nombreux avantages pour l'identification de pathogènes, mais ont besoin d'être améliorées pour la détection d'un plus large panel de résistances aux antibiotiques.