Exposing and Overcoming Limitations of Clinical Laboratory Tests in COVID-19 by Adding Immunological Parameters; A Retrospective Cohort Analysis and Pilot Study

BackgroundTwo years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted for clinical management and in most algorithms the contribution of laboratory variables is limited. ObjectivesTo measure the predictive performance of currently used clinical laboratory tests alone or combined with clinical variables and explore the predictive power of immunological tests adequate for clinical laboratories. Methods: Data from 2,600 COVID-19 patients of the first wave of the pandemic in the Barcelona area (exploratory cohort of 1,579, validation cohorts of 598 and 423 patients) including clinical parameters and laboratory tests were retrospectively collected. 28-day survival and maximal severity were the main outcomes considered in the multiparametric classical and machine learning statistical analysis. A pilot study was conducted in two subgroups (n=74 and n=41) measuring 17 cytokines and 27 lymphocyte phenotypes respectively. Findings1) Despite a strong association of clinical and laboratory variables with the outcomes in classical pairwise analysis, the contribution of laboratory tests to the combined prediction power was limited by redundancy. Laboratory variables reflected only two types of processes: inflammation and organ damage but none reflected the immune response, one major determinant of prognosis. 2) Eight of the thirty variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the combined statistical predictive power. 3) The interpretation of clinical and laboratory variables was moderately improved by grouping them in two categories i.e., inflammation related biomarkers and organ damage related biomarkers; Age and organ damage-related biomarker tests were the best predictors of survival, and inflammatory-related ones were the best predictors of severity. 4) The pilot study identified immunological tests (CXCL10, IL-6, IL-1RA and CCL2), that performed better than most currently used laboratory tests. ConclusionsLaboratory tests for clinical management of COVID 19 patients are valuable but limited predictors due to redundancy; this limitation could be overcome by adding immunological tests with independent predictive power. Understanding the limitations of tests in use would improve their interpretation and simplify clinical management but a systematic search for better immunological biomarkers is urgent and feasible

Apoptosis, toll-like, RIG-I-like and NOD-like receptors are pathways jointly induced by diverse respiratory bacterial and viral pathogens

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.The authors gratefully acknowledge financial support from the “CIBER de Enfermedades Respiratorias” (CIBERES), an initiative of the “Instituto de Salud Carlos III” (ISCIII), Spain. Research activities in the participating laboratories received further funding from the following sources: Centro Nacional de Microbiología, ISCIII, PI15CIII/00024 and MINECO (SAF2015- 67033-R); Centro Nacional de Biotecnología, MINECO (BFU2014-57797-R); Hospital Universitari Germans Trias I Pujol, Spanish Society of Pneumology and Thoracic Surgery (SEPAR 054/2011); Departamento de Bioquímica y Biología Molecular I, MINECO (SAF2015-65307-R); Centro de Investigaciones Biológicas, MINECO (SAF2012-39444-C01/02); Fundación de Investigación Sanitaria de las Islas Baleares, MINECO (SAF2012-39841); Instituto de Agrobiotecnología, MINECO (SAF2015-66520-R); Instituto de Química Física Rocasolano, MINECO (BFU2015-70052-R) and the Marie Curie Initial Training Network GLYCOPHARM (PITN-GA- 2012-317297). Subprograma Estatal de Formación (BES-2013- 065355)

Estrategias de Klebsiella Pneumoniae para subvertir los sistemas defensivos del huésped

[spa] Klebsiella pneumoniae (KP) es uno de los principales patógenos nosocomiales y un notable causante de infecciones adquiridas en la comunidad, hechos que suponen un importante problema de salud pública. Además, en los últimos años está aumentando constantemente el número de aislados de KP resistentes a antibióticos, lo cual agrava la problemática clínico-económica asociada a estas infecciones. Por eso, necesitamos desarrollar nuevas estrategias terapéuticas contra KP. Así, aparte de nuevos fármacos destinados a tener una actividad bacteriostática/bactericida directa, encontrar dianas para optimizar la respuesta inmune del huésped contra KP, es un campo de estudio que se debe expandir. Para ello es necesario conocer mejor el proceso de infección por KP y los factores de virulencia que usa la bacteria para llevarla a cabo. Del mismo modo, es importante conocer las vías de señalización que se activan en las células del huésped durante la infección, ya que la subversión de la inflamación y de la inmunidad innata son esenciales para la supervivencia de KP y la progresión de la infección. Ganar conocimiento acerca de estos procesos podría suponer una fuente de dianas terapéuticas eficaces para el desarrollo de fármacos contra KP. El calcio libre (Ca2+) y el AMP cíclico son dos importantes segundos mensajeros implicados en gran variedad de procesos celulares. En algunas infecciones bacterianas, la señalización dependiente de ellos está involucrada en la secreción de mediadores pro-inflamatorios. Otro importante facilitador de la inmunidad innata es la familia de los interferones, que una vez segregados al medio y detectados por las células, ponen en marcha rutas de señalización a la postre inflamatorias, importantes no solamente durante infecciones víricas sino también bacterianas. La relación de estos mensajeros y vías con el regulador transcripcional NF-κB, que en última instancia gobierna la expresión de genes pro-inflamatorios es muy estrecha y bien conocida. Sin embargo, estos campos no están apenas estudiados en relación al caso concreto de las infecciones por KP. En esta tesis se han estudiado en profundidad estas vías de señalización, en concreto en relación a la atenuación de la respuesta inflamatoria del huésped, activamente promovida por KP como una de sus principales estrategias para tener éxito en la infección. Nuestros resultados muestran que KP induce un aumento del Ca2+ intracelular libre procedente de su liberación del retículo endoplásmico (RE) en células pulmonares humanas. Esta señalización se lleva a cabo principalmente a través de la activación de TLR1/2, aunque también participa la activación de TLR4, y para ella son imprescindibles la presencia de Ca2+ extracelular, la viabilidad de KP y ciertos factores de virulencia, entre los que destaca especialmente la cápsula. La detección de KP por parte estos receptores parece causar la fosforilación de PLCγ, de la cual se deriva la consiguiente y mencionada liberación del Ca2+ desde el RE, que desemboca en el incremento del AMP cíclico. Este incremento conduciría a la presumible fosforilación del regulador CREB, que compite con NF-κB por la promoción de la expresión de los respectivos genes bajo su control, finalmente consiguiendo una atenuación de la respuesta inflamatoria. Por otro lado, nuestros resultados sugieren que KP inhibe activamente la fosforilación de STAT1, bloqueando así la señalización inflamatoria dependiente de IFNs (por una vía ajena a las proteínas SOCS1, SOCS3 y SHP2), para lo cual es esencial la viabilidad bacteriana. Los resultados presentados en esta tesis nos permiten proponer un novedoso modelo de atenuación inflamatoria promovida activamente por parte de KP, revelando nuevas estrategias que sigue esta especie para subvertir la respuesta inmune del hospedador, y que destacan por su complejidad al afectar a diferentes rutas de señalización (Ca2+, AMP cíclico e IFNs-dependientes).[cat] Klebsiella penumoniae (KP) és un dels principals patògens nosocomials i un notable agent causant d’infeccions adquirides en la comunitat, fets que suposen un important problema de salut pública. A més, en els darrers anys està augmentant constantment el nombre d’aïllats de KP resistents als antibiòtics, cosa que agreuja la problemàtica clínica-econòmica associada a aquestes infeccions. Per això, necessitem desenvolupar noves estratègies terapèutiques contra KP. Així, apart de nous fàrmacs destinats a tenir una activitat bacteriostàtica/bactericida directa, trobar dianes per optimitzar la resposta immune de l’hoste contra KP, és un camp d’estudi que s’ha d’expandir. Per això és necessari conèixer millor el procés d’infecció per KP i els factors de virulència que usa la bactèria per dur-la a terme. Igualment, és important conèixer les vies de senyalització activades en les cèl·lules de l’hoste durant la infecció, ja que la subversió de la inflamació i de la immunitat innata són essencials per a la supervivència de KP i la progressió de la infecció. Guanyar coneixement sobre aquests processos suposaria una font de dianes terapèutiques eficaces per el desenvolupament de fàrmacs contra KP. El calci lliure (Ca2+) i l’AMP cíclic són dos importants segons missatgers implicats en gran varietat de processos cel·lulars. En algunes infeccions bacterianes, la senyalització dependent d’ells està involucrada en la secreció de mediadors proinflamatoris. Un altre facilitador de la immunitat innata és la família dels interferons, que una vegada secretats i detectats per les cèl·lules, posen en marxa rutes de senyalització al cap i a la fi inflamatòries, importants no només durant infeccions víriques sinó també bacterianes. La relació d’aquests missatgers i vies amb el regulador transcripcional NF-κB, que en darrera instància governa la expressió de gens pro-inflamatoris és molt estreta i ben coneguda. Malgrat això, aquests camps no estan quasi estudiats en relació al cas concret de les infeccions per KP. En aquesta tesi s’han estudiat en profunditat aquestes vies de senyalització, en concret en relació a la atenuació de la resposta inflamatòria de l’hoste, activament promoguda per KP com una de les seves principals estratègies per tenir èxit a la infecció. Els nostres resultats mostren que KP indueix un augment del Ca2+ intracel·lular lliure procedent de la seva alliberació del reticle endoplàsmic (RE) en cèl·lules pulmonars humanes. Aquesta senyalització es porta a terme principalment a través de la activació de TLR1/2, malgrat també hi participa la activació de TLR4, i per ella són imprescindibles la presencia de Ca2+ extracel·lular, la viabilitat de KP i certs factors de virulència, entre els que destaca especialment la càpsula. La detecció de KP per part d’aquests receptors sembla causar la fosforil·lació de PLCγ, de la qual es deriva la conseqüent i mencionada alliberació del Ca2+ des del RE, que desemboca en l’increment de l’AMP cíclic. Aquest increment conduiria a la presumible fosforil·lació del regulador CREB, que competeix amb NF-κB per la promoció de la expressió dels seus respectius gens baix el seu control, finalment aconseguint una atenuació de la resposta inflamatòria. Per altra banda, els nostres resultats suggereixen que KP inhibeix activament la fosforil·lació de STAT1, bloquejant així la senyalització inflamatòria depenent de IFNs (per una via aliena a les proteïnes SOCS1, SOCS3 i SHP2), per lo qual és essencial la viabilitat bacteriana. Els resultats presentats en aquesta tesi ens permeten proposar un nou model d’atenuació inflamatòria promoguda activament per part de KP, revelant noves estratègies que segueix aquesta espècie per subvertir la resposta immune de l’hoste, i que destaquen per la seva complexitat al afectar a diferents rutes de senyalització (Ca2+, AMP cíclic i IFNs-dependents).[eng] Klebsiella pneumoniae (KP) is one of the main nosocomial opportunistic pathogens as well as a notable cause of community-acquired infections, facts that pose an important problem of public health. Additionally, during the last years, the number of antibiotic-resistant KP isolates is constantly increasing, facts that aggravate the clinical-economical implications associated with the infection by this species. It is obvious, therefore, that we need to develop new therapeutic strategies against KP. Thus, besides new drugs intended to have direct bacteriostatic/bactericidal activity, finding targets to improve the immune response of the host against KP, is a research field that needs to be expanded. Therefore, to deeply understand the KP infection process and the virulence factors used by KP, is urgently needed. Similarly, it is important to dissect the signaling pathways activated in the host cells during infection, since the subversion of the inflammation and the innate immunity are essential strategies used by KP for the progression of the infection. To acquire knowledge about these processes could be a decisive source of effective therapeutic targets for the future development of drugs against KP. The free calcium (Ca2+) and the cyclic AMP are two important second messengers involved in a wide variety of cellular processes. In some bacterial infections, several signaling pathways strongly depending on them ultimately promote the secretion of inflammatory mediators. Another important stimulator of the innate immunity is the interferon family, which once secreted to the medium and detected by the cells, start signaling pathways that ultimately are inflammatory, important not only during viral but also bacterial infections. The relation of these messengers and pathways with the NF-κB transcriptional regulator, which controls the expression of certain proinflammatory genes, is tight and well-known. However, these fields are poorly studied regarding the specific case of KP infections. In this thesis, we deeply studied these signaling pathways, specifically about the attenuation of the inflammatory response in the host, actively induced by KP as one of their main strategies to succeed. Our results show that KP induces an increase in intracellular free Ca2+ through its release from the endoplasmic reticulum (ER) in human lung cells. This signaling is carried out mainly through the activation of TLR1/2, although TLR4 also takes part in the process, , for which the presence of extracellular Ca2+, the KP viability, and some virulence factors (being the capsule probably the most important), are required. The detection of KP by these receptors seems to cause the phosphorylation of PLCγ , that triggers the Ca2+ release from the ER, which ultimately causes the increase of cyclic AMP. This increase would drive to the presumable phosphorylation of CREB, which competes with NF-κB for the promotion of the expression of their respective controlled genes, causing a final mitigation of the inflammatory response. On the other hand, our results suggest that KP actively inhibits the phosphorylation of STAT1, therefore blocking the IFNs-dependent inflammatory signaling (through a pathway independent of the proteins SOCS1, SOCS3, and SHP2), process for which the bacterial viability is essential. The results presented in this thesis allow us to propose a new model of attenuation of the inflammatory response actively promoted by KP, revealing new strategies that this species follow to subvert the host immune response, and that stand out due to their complexity, since they affect different signaling pathways (Ca2+, cyclic AMP, and IFNs-dependent)

Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling

Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia

Apoptosis, Toll-like, RIG-I-like and NOD-like receptors are pathways jointly induced by diverse respiratory bacterial and viral pathogens

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here

Exposing and Overcoming Limitations of clinical laboratory tests in COVID-19 by adding immunological parameters; A Retrospective cohort analysis and pilot study

Background: Two years since the onset of the COVID-19 pandemic no predictive algorithm has been generally adopted for clinical management and in most algorithms the contribution of laboratory variables is limited. Objectives: To measure the predictive performance of currently used clinical laboratory tests alone or combined with clinical variables and explore the predictive power of immunological tests adequate for clinical laboratories. Methods: Data from 2,600 COVID-19 patients of the first wave of the pandemic in the Barcelona area (exploratory cohort of 1,579, validation cohorts of 598 and 423 patients) including clinical parameters and laboratory tests were retrospectively collected. 28-day survival and maximal severity were the main outcomes considered in the multiparametric classical and machine learning statistical analysis. A pilot study was conducted in two subgroups (n=74 and n=41) measuring 17 cytokines and 27 lymphocyte phenotypes respectively. Findings: 1) Despite a strong association of clinical and laboratory variables with the outcomes in classical pairwise analysis, the contribution of laboratory tests to the combined prediction power was limited by redundancy. Laboratory variables reflected only two types of processes: inflammation and organ damage but none reflected the immune response, one major determinant of prognosis. 2) Eight of the thirty variables: age, comorbidity index, oxygen saturation to fraction of inspired oxygen ratio, neutrophil-lymphocyte ratio, C-reactive protein, aspartate aminotransferase/alanine aminotransferase ratio, fibrinogen, and glomerular filtration rate captured most of the combined statistical predictive power. 3) The interpretation of clinical and laboratory variables was moderately improved by grouping them in two categories i.e., inflammation related biomarkers and organ damage related biomarkers; Age and organ damage-related biomarker tests were the best predictors of survival, and inflammatory-related ones were the best predictors of severity. 4) The pilot study identified immunological tests (CXCL10, IL-6, IL-1RA and CCL2), that performed better than most currently used laboratory tests. Conclusions: Laboratory tests for clinical management of COVID 19 patients are valuable but limited predictors due to redundancy; this limitation could be overcome by adding immunological tests with independent predictive power. Understanding the limitations of tests in use would improve their interpretation and simplify clinical management but a systematic search for better immunological biomarkers is urgent and feasible

Ticagrelor in patients with diabetes and stable coronary artery disease with a history of previous percutaneous coronary intervention (THEMIS-PCI) : a phase 3, placebo-controlled, randomised trial

Background: Patients with stable coronary artery disease and diabetes with previous percutaneous coronary intervention (PCI), particularly those with previous stenting, are at high risk of ischaemic events. These patients are generally treated with aspirin. In this trial, we aimed to investigate if these patients would benefit from treatment with aspirin plus ticagrelor. Methods: The Effect of Ticagrelor on Health Outcomes in diabEtes Mellitus patients Intervention Study (THEMIS) was a phase 3 randomised, double-blinded, placebo-controlled trial, done in 1315 sites in 42 countries. Patients were eligible if 50 years or older, with type 2 diabetes, receiving anti-hyperglycaemic drugs for at least 6 months, with stable coronary artery disease, and one of three other mutually non-exclusive criteria: a history of previous PCI or of coronary artery bypass grafting, or documentation of angiographic stenosis of 50% or more in at least one coronary artery. Eligible patients were randomly assigned (1:1) to either ticagrelor or placebo, by use of an interactive voice-response or web-response system. The THEMIS-PCI trial comprised a prespecified subgroup of patients with previous PCI. The primary efficacy outcome was a composite of cardiovascular death, myocardial infarction, or stroke (measured in the intention-to-treat population). Findings: Between Feb 17, 2014, and May 24, 2016, 11 154 patients (58% of the overall THEMIS trial) with a history of previous PCI were enrolled in the THEMIS-PCI trial. Median follow-up was 3·3 years (IQR 2·8–3·8). In the previous PCI group, fewer patients receiving ticagrelor had a primary efficacy outcome event than in the placebo group (404 [7·3%] of 5558 vs 480 [8·6%] of 5596; HR 0·85 [95% CI 0·74–0·97], p=0·013). The same effect was not observed in patients without PCI (p=0·76, p interaction=0·16). The proportion of patients with cardiovascular death was similar in both treatment groups (174 [3·1%] with ticagrelor vs 183 (3·3%) with placebo; HR 0·96 [95% CI 0·78–1·18], p=0·68), as well as all-cause death (282 [5·1%] vs 323 [5·8%]; 0·88 [0·75–1·03], p=0·11). TIMI major bleeding occurred in 111 (2·0%) of 5536 patients receiving ticagrelor and 62 (1·1%) of 5564 patients receiving placebo (HR 2·03 [95% CI 1·48–2·76], p<0·0001), and fatal bleeding in 6 (0·1%) of 5536 patients with ticagrelor and 6 (0·1%) of 5564 with placebo (1·13 [0·36–3·50], p=0·83). Intracranial haemorrhage occurred in 33 (0·6%) and 31 (0·6%) patients (1·21 [0·74–1·97], p=0·45). Ticagrelor improved net clinical benefit: 519/5558 (9·3%) versus 617/5596 (11·0%), HR=0·85, 95% CI 0·75–0·95, p=0·005, in contrast to patients without PCI where it did not, p interaction=0·012. Benefit was present irrespective of time from most recent PCI. Interpretation: In patients with diabetes, stable coronary artery disease, and previous PCI, ticagrelor added to aspirin reduced cardiovascular death, myocardial infarction, and stroke, although with increased major bleeding. In that large, easily identified population, ticagrelor provided a favourable net clinical benefit (more than in patients without history of PCI). This effect shows that long-term therapy with ticagrelor in addition to aspirin should be considered in patients with diabetes and a history of PCI who have tolerated antiplatelet therapy, have high ischaemic risk, and low bleeding risk