Importància diagnòstica i pronòstica dels marcadors d'inflamació en patalogia infecciosa : noves molècules antiinflamatòries bacterianes /

Títol obtingut de la portada digitalitzadaConsultable des del TDXLa resposta inflamatòria consisteix en l'activació de mecanismes humorals i cel·lulars en resposta a un estímul, amb l'objectiu de limitar-ne els potencials efectes nocius. Quan l'estímul que desencadena la resposta és la presència d'un microorganisme viable o bé els seus productes, els mecanismes de defensa poden autolimitar-ne els efectes i confinar la infecció a un òrgan o fins i tot eradicar-la o bé, si falla aquesta contenció, pot persistir i evolucionar donant lloc a l'alliberament de múltiples mitjancers al torrent circulatori, produint una resposta inflamatòria sistèmica i fallida orgànica. En la inflamació intervenen factors dependents del microorganisme i factors dependents de l'hostatger, donant lloc a les diverses manifestacions clíniques. Disposar de marcadors que permetin mesurar el grau de resposta inflamatòria ha de permetre instaurar les mesures terapèutiques més adequades a cada situació, amb especial èmfasi al desenvolupament de tractaments immunomoduladors, actuant de forma complementària a l'administració d'agents antimicrobians i mesures de suport. Els objectius d'aquesta Tesi han estat, d'una banda, avaluar la utilitat de la determinació de diversos marcadors d'inflamació per al diagnòstic i monitorització de diverses síndromes clíniques d'origen infecciós. D'altra banda, des del punt de vista del microorganisme, es descriuen dues noves molècules amb funcions antiinflamatòries d'origen estafilocòccic, que actuen interferint en la resposta immunitària innata. Els resultats mostren que els marcadors reflecteixen el grau de resposta inflamatòria i varien en funció de l'etiologia, l'edat, la severitat i el temps d'evolució de la infecció. Cal contextualitzar la utilitat de cada marcador a la localització de la infecció, la situació clínica i la presència de comorbilitats. La procalcitonina (PCT) es mostra superior a la proteïna C reactiva (PCR) per distingir entre infecció bacteriana i infecció vírica, i també entre infeccions bacterianes i causes no infeccioses d'inflamació, aportant informació sobre l'etiologia però també sobre la resposta inflamatòria de l'hostatger. Com a limitacions, la PCT no augmenta, o només discretament, quan la infecció es troba confinada a un òrgan, sense manifestacions sistèmiques. En aquesta situació, la PCR aportaria més informació. Tampoc no és útil en la monitorització de les infeccions víriques. En aquests casos, la neopterina i/o la PCR podrien resultar complementàries. L'ús de marcadors no ha de substituir mai un correcte diagnòstic i seguiment clínic i microbiològic, però pot aportar una més completa informació diagnòstica i pronòstica obrint fins i tot noves opcions terapèutiques quant a la utilització de tractaments immunomoduladors. És probable que l'òptima rendibilitat s'obtingui amb l'adequada combinació de marcadors, tenint en compte els avantatges i limitacions de cadascun, a més de la utilització de determinacions seriades en funció de la seva cinètica. D'altra banda s'han caracteritzat dues noves molècules estafilocòcciques que presenten cert grau d'homologia amb la prèviament descrita CHIPS (Chemotaxis inhibitory protein of Staphylococcus aureus). Els resultats obtinguts mostren que la proteïna excretada FPR-like 1 Inhibitory protein (FLIPr) actua bloquejant el receptor de pèptids formilats FPRL1. La troballa de FLIPr com a antagonista d'aquest receptor és de gran importància clínica per la seva potencial intervenció en la patogènesi de la malaltia estafilocòccica. FPRL1 és, a més, el receptor de diverses molècules implicades en la resposta inflamatòria i en processos patològics de la importància de l'amiloidosi sistèmica, la malaltia d'Alzheimer o la malaltia priònica. La segona proteïna, que s'ha anomenat FLIPr-like, actua bloquejant tant FPR com FPRL1. El millor coneixement dels mecanismes d'evasió del sistema immunitari a càrrec de microorganismes prototipus com Staphylococcus aureus ha de permetre un també millor coneixement dels fonaments de la patogenicitat bacteriana, la immunitat innata i les possibles vies d'intervenció. L'estudi coordinat de factors dependents del microorganisme i la resposta inflamatòria generada per l'hostatger ha de donar lloc a la major comprensió de les complexes interaccions entre ambdós que donen lloc a la malaltia infecciosa i en condicionen les seves manifestacions i pronòstic.Inflammatory response involves the activation of multiple humoral and cellular mechanisms in response to a stimulus, with the objective of limiting its noxious effects. When the inflammatory response is stimulated by a viable microorganism or its products, the immune system may limit its effects and confine the infection to an organ or even eradicate it. If the contention falls, the response may persist and evolve to systemic inflammation and organ failure by the liberation of multiple mediators to the circulatory system. Inflammatory response involves factors depending of the microorganism and factors depending of the host, leading to several clinical symptoms. The measurement of biomarkers should allow to measure the degree of inflammatory response. The knowledge of the inflammatory «status» should allow the development and use of immunomodulatory therapies, complementary to antimicrobial agents and supportive therapy. The aims of this Thesis were, first of all, to evaluate the utility of the measurement of several inflammatory mediators for the diagnosis and follow-up of several infectious clinical syndromes. On the other hand, from the point of view of the microorganism, two new antiinflamatory molecules secreted by Staphylococcus aureus are described. Both molecules interfere with innate immune response. The results show that the evaluated biomarkers reflect the degree of inflammatory response and vary depending on etiology, age, severity and time of evolution of the infection. The utility of every biomarker has to be contextualized depending on the localization of infection, the clinical setting and the presence of comorbidities. Procalcitonin (PCT) shows better sensitivity and specificity than C reactive protein (CRP) in distinguishing between bacterial and viral infection and also between bacterial infection and inflammation of non infectious origin, giving information about the inflammatory response of the host. As limitations, PCT does not increase, or only slightly, when the infection is confined to an organ, without systemic involvement. In this situation, CRP may give more information. PCT is also not useful in the follow-up of viral infections. In those cases, CRP and/or neopterin may be complementary. The use of inflammatory markers does not aim to substitute an adequate clinical and microbiological diagnosis but may offer a more complete diagnostic and prognostic information, leading to the development of new therapeutical options such as immunomodulatory treatments. The optimal reliability may be obtained by the right combination of biomarkers, taking in account the advantages and limitations of each. The measurement of seriated samples according to the kinetics of every biomarker is also useful. On the other hand, two new staphylococcal molecules have been characterized. They both have some homology with the previously described CHIPS (Chemotaxis inhibitory protein of Staphylococcus aureus). The obtained results show that the excreted protein FPR-like 1 Inhibitory protein (FLIPr) acts blockading the formylated peptide receptor FPRL1. The finding of FLIPr as an antagonist of this receptor is of clinical importance because of its potential intervention in the pathogenesis of staphylococcal diseases. Furthermore, FPRL1 is the receptor of several molecules involved in the inflammatory response and in pathological processes as important as systemic amiloidosis, Alzheimer disease or prion diseases. The second protein, that has been called FLIPr-like, acts blockading both FPR and FPRL1. The better knowledge of immune evasion mechanisms by prototype microorganisms such as Staphylococcus aureus has to allow a better knowledge of the basis of bacterial pathogenicity, innate immunity and the potential ways of intervention. The coordinate study of factors depending of the microorganism and the inflammatory response generated by the host should lead to a better understanding of the complex interactions between a pathogen and its host, that lead to an infectious disease and condition its clinical presentation and prognosis

Cigarette smoke exposure redirects Staphylococcus aureus to a virulence profile associated with persistent infection

Altres ajuts: This work also received a grant from the Spanish Society of Pneumology and Thoracic Surgery (SEPAR 024/2016). M.L. gratefully acknowledges funding from the European Respiratory Society Long-term Fellowship grant (LTRF-5934). A.M.E. acknowledges funding from the Royal Society, Department of Medicine (Imperial College), and from the Imperial NIHR Biomedical Research Centre, Imperial College London. B.C.Y is funded by an NIHR Clinical Lectureship. CP acknowledges Programa Germans Trias Sapiens Fundació Catalunya la Pedrera and European Respiratory Society short term research fellowship October 2018 (STRTF201810-00467).Tobacco smoking represents the leading preventable cause of death worldwide. Smoking is a recognised risk factor for several pathologies and is detrimental to host immune surveillance and defence. However, the impact of smoking on microbial residents of the nasopharyngeal cavity, in contact with cigarette smoke (CS), is lacking. Staphylococcus aureus is a major human pathogen that colonises the human nasopharynx and causes a wide range of infections. We investigated the impact of CS on specific virulence phenotypes important in S aureus pathogenesis. We observed strain-dependent differences following exposure to CS, namely growth inhibition, augmented biofilm formation, increased invasion of, and persistence within, bronchial alveolar epithelial cells. Additionally, we confirm the critical role of a functional accessory gene regulator (Agr) system in mediating increased biofilm development and host cell invasion and persistence following CS exposure. Furthermore, CS exposure resulted in reduced toxin production. Importantly, exposure of S aureus to CS accelerated the frequency of mutations and resulted in a significant increase in gentamicin-resistant small colony variant (SCV) formation. Mutational analysis revealed that CS induced SCVs emerge via the SOS response DNA mutagenic repair system. Taken together, our results suggest that CS redirects certain S aureus strains to a virulence profile associated with persistence

E-cigarettes : effects in phagocytosis and cytokines response against Mycobacterium tuberculosis

Cigarette smoking and tuberculosis are a significant cause of death worldwide. Several epidemiological studies have demonstrated cigarette smoking is a risk factor for tuberculosis. Electronic cigarettes have recently appeared as a healthier alternative to conventional smoking, although their impact in tuberculosis is not well understood. The aim of this study was to explore the effect of electronic cigarettes in phagocytosis of Mycobacterium tuberculosis and cytokines production. In vitro infection was carried out by exposing THP-1 macrophages to four electronic vapor extracts and the intracellular burden of M. tuberculosis was determined. The percentage of infection was evaluated by confocal microscopy and the cytokine production by Luminex. A reduction of intracellular M. tuberculosis burden in THP- 1 macrophages was found after its exposure to electronic vapor extract; the same trend was observed by confocal microscopy when Mycobacterium bovis BCG-GFP strain was used. Electronic cigarettes stimulate a pro-inflammatory cytokine response. We conclude that electronic cigarettes impair the phagocytic function and the cytokine response to M. tuberculosis

Advances in diagnostic tools for respiratory tract infections : from tuberculosis to COVID-19 - changing paradigms?

Respiratory tract infections (RTIs) are one of the most common reasons for seeking healthcare, but are amongst the most challenging diseases in terms of clinical decision-making. Proper and timely diagnosis is critical in order to optimise management and prevent further emergence of antimicrobial resistance by misuse or overuse of antibiotics. Diagnostic tools for RTIs include those involving syndromic and aetiological diagnosis: from clinical and radiological features to laboratory methods targeting both pathogen detection and host biomarkers, as well as their combinations in terms of clinical algorithms. They also include tools for predicting severity and monitoring treatment response. Unprecedented milestones have been achieved in the context of the COVID-19 pandemic, involving the most recent applications of diagnostic technologies both at genotypic and phenotypic level, which have changed paradigms in infectious respiratory diseases in terms of why, how and where diagnostics are performed. The aim of this review is to discuss advances in diagnostic tools that impact clinical decision-making, surveillance and follow-up of RTIs and tuberculosis. If properly harnessed, recent advances in diagnostic technologies, including omics and digital transformation, emerge as an unprecedented opportunity to tackle ongoing and future epidemics while handling antimicrobial resistance from a One Health perspective. Unprecedented advances have been achieved by improving and applying the latest technologies to key aspects such as epidemiology, contact tracing and diagnostics of respiratory tract infections that need to be properly addressed for global improvement

Molecular detection of mycobacterium tuberculosis in oral mucosa from patients with presumptive tuberculosis

Funding: This research was funded by a Strategic Award grant from the European and Developing Countries Clinical Trials Partnership (grant DRIA2014-309) and its cofounders, the Medical Research Council UK, and Institutode Salud Carlos III (ISCIII), Spain (PI116/01912); and from the European Union’s Horizon 2020 Research and Innovation Programme under the Marie Skłodowska-Curie grant agreement no. 823854 (INNOVA4TB).Tuberculosis (TB) diagnosis is increasingly based on the detection of Mycobacterium tuberculosis complex (MTBC) DNA in sputum using molecular diagnostic tests as the first test for diagnosis. However, sputum can be difficult to obtain in children, patients without productive cough, and the elderly and approaches testing non-sputum samples are needed. We evaluated whether TB can be detected from the oral mucosa of patients with TB. Adults with presumptive TB were examined using culture, Xpert MTB/RIF, smear microscopy and X-Rays. Oral mucosa swabs collected on PrimeStore-MTM, stored at room temperature if tested within 30 days or at −20 °C if examined at a later time. RT-PCR was performed to detect M. tuberculosis DNA. Eighty patients had bacteriologically-confirmed TB, 34 had bacteriologically-negative TB (negative tests but abnormal X-rays) and 152 were considered not to have TB (not TB). Oral swabs RT-PCR were positive in 29/80 (36.3%) bacteriologically-confirmed, 9/34 (26.5%) bacteriologically-negative and 29/152 (19.1%) not TB. The yield varied among samples stored for less and more than 30 days (p = 0.013) from 61% (11/18) and 29% (18/62) among bacteriologically confirmed, and 30.8% (4/13) and 23.8% (5/21) among bacteriologically-negative participants. Among not TB patients, the specificity was 80.9% (123/152), being 78.3% (18/23) among samples stored less than 30 days and 81.4% (105/129) among samples stored for more than 30 days (p = 0.46). The detection of M. tuberculosis in oral mucosa samples is feasible, but storage conditions may affect the yield.Publisher PDFPeer reviewe

Genotypic and Phenotypic Characterization of Staphylococcus aureus Isolates from the Respiratory Tract in Mechanically-Ventilated Patients

Staphylococcus aureus is a commensal and frequent colonizer of the upper respiratory tract. When mechanical ventilation disrupts natural defenses, S. aureus is frequently isolated from the lower airways, but distinguishing between colonization and infection is difficult. The objectives of this study were (1) to investigate the bacterial genome sequence in consecutive isolates in order to identify changes related to the pathological adaptation to the lower respiratory tract and (2) to explore the relationship between specific phenotypic and genotypic features with the patient's study group, persistence of the clinical isolate and clinical outcome. A set of 94 clinical isolates were selected and corresponded to 34 patients that were classified as having pneumonia (10), tracheobronchitis (11) and bronchial colonization (13). Clinical strains were phenotypically characterized by conventional identification and susceptibility testing methods. Isolates underwent whole genome sequencing using Illumina HiSeq4000. Genotypic characterization was performed with an in-house pipeline (BacterialTyper). Genomic variation arising within-host was determined by comparing mapped sequences and de novo assemblies. Virulence factors important in staphylococcal colonization and infection were characterized using previously established functional assays. (1) Toxin production was assessed using a THP-1 cytotoxicity assay, which reports on the gross cytotoxicity of individual isolates. In addition, we investigated the expression of the major virulence factor, alpha-toxin (Hla) by Western blot. (2) Adhesion to the important extracellular matrix molecule, fibronectin, was determined using a standardized microtitre plate assay. Finally, invasion experiments using THP-1 and A539 cell lines and selected clinical strains were also performed. Repeated isolation of S. aureus from endotracheal aspirate usually reflects persistence of the same strain. Within-host variation is detectable in this setting, but it shows no evidence of pathological adaptation related to virulence, resistance or niche adaptations. Cytotoxicity was variable among isolates with 14 strains showing no cytotoxicity, with these latter presenting an unaltered Fn binding capacity. No changes on cytotoxicity were reported when comparing study groups. Fn binding capacity was reported for almost all strains, with the exception of two strains that presented the lowest values. Strains isolated from patients with pneumonia presented a lower capacity of adhesion in comparison to those isolated during tracheobronchitis (p = 0.002). Hla was detected in 71 strains (75.5%), with most of the producer strains in pneumonia and bronchial colonization group (p = 0.06). In our cohort, Hla expression (presence or absence) in sequential isolates was usually preserved (70%) although in seven cases the expression varied over time. No relationship was found between low cytotoxicity and intracellular persistence in invasion experiments. In our study population, persistent S. aureus isolation from airways in ventilated patients does not reflect pathological adaptation. There is an important diversity of sequence types. Cytotoxicity is variable among strains, but no association with study groups was found, whereas isolates from patients with pneumonia had lower adhesion capability. Favorable clinical outcome correlated with increased bacterial adhesion in vitro. Most of the strains isolated from the lower airways were Hla producers and no correlation with an adverse outcome was reported. The identification of microbial factors that contribute to virulence is relevant to optimize patient management during lower respiratory tract infections

Use of IP-10 detection in dried plasma spots for latent tuberculosis infection diagnosis in contacts via mail

The aim of this study was to test the use of IP-10 detection in dried plasma from contact studies individuals (contacts of smear positive patients), by comparing it with IP-10 and IFN-γ detection in direct plasma, to establish IP-10 detection in DPS as a useful assay for LTBI diagnosis. Whole blood samples were collected from 80 subjects: 12 with active tuberculosis (TB), and 68 from contact studies. The amount of IFN-γ produced by sensitized T cells was determined in direct plasma by QuantiFERON Gold In-Tube test. IP-10 levels were determined in direct and dried plasma by an in-house ELISA. For dried plasma IP-10 determination, two 25 µl plasma drops were dried in Whatman903 filter paper and sent by mail to the laboratory. Regarding TB patients, 100.0%, 91.7% and 75.0% were positive for IFN-γ detection and IP-10 detection in direct and dried plasma, respectively. In contacts, 69.1%, 60.3% and 48.5% had positive results after IFN-γ and IP-10 in direct and dried plasma, respectively. The agreement among in vitro tests was substantial and IP-10 levels in direct and dried plasma were strongly correlated (r = 0.897). In conclusion, IP-10 detection in dried plasma is a simple and safe method that would help improve LTBI management

Acute phase proteins and IP-10 in plasma for tuberculosis diagnosis

Background: Tuberculosis (TB) is a leading cause of death from a single infectious agent, and triage tests based on biomarkers may help to improve the diagnosis. This study aims to determine whether C-reactive protein (CRP), interferon-γ-inducible protein 10 (IP-10), α1-acid glycoprotein (AGP), and α1-anti-trypsin (AAT) could be useful for a screening test in patients with presumptive TB disease. Methods: CRP, IP-10, AGP, and AAT were measured in plasma samples from 277 patients with presumptive TB disease in the Republic of Moldova in a prospective study. Results: In general, the levels of all the biomarkers were higher in patients with TB than in the other groups (p < 0.05). Receiver operating characteristic curve analyses showed an area under the curve lower than 0.7 for all the biomarkers, and low correlations (Spearman's r < 0.6) were found between biomarkers. Conclusion: The levels of the tested biomarkers were different throughout the patient groups studied, but their suboptimal diagnostic performance either as individual biomarkers or in combination does not favor their use for triage testing

Immunogenicity of 60 novel latency-related antigens of Mycobacterium tuberculosis

The aim of our work here was to evaluate the immunogenicity of 60 mycobacterial antigens, some of which have not been previously assessed, notably a novel series of in vivo -expressed Mycobacterium tuberculosis (IVE-TB) antigens. We enrolled 505 subjects and separated them in individuals with and without latent tuberculosis infection (LTBI) vs. patients with active tuberculosis (TB). Following an overnight and 7 days stimulation of whole blood with purified recombinant M. tuberculosis antigens, interferon-γ (IFN-γ) levels were determined by ELISA. Several antigens could statistically significantly differentiate the groups of individuals. We obtained promising antigens from all studied antigen groups [dormancy survival regulon (DosR regulon) encoded antigens; resuscitation-promoting factors (Rpf) antigens; IVE-TB antigens; reactivation associated antigens]. Rv1733, which is a probable conserved transmembrane protein encoded in DosR regulon, turned out to be very immunogenic and able to discriminate between the three defined TB status, thus considered a candidate biomarker. Rv2389 and Rv2435n, belonging to Rpf family and IVE-TB group of antigens, respectively, also stood out as LTBI biomarkers. Although more studies are needed to support our findings, the combined use of these antigens would be an interesting approach to TB immunodiagnosis candidates

Accessory gene regulator (Agr) functionality in Staphylococcus aureus derived from lower respiratory tract infections

Altres ajuts: This work has been funded by the project PI13/01418 which is part of "Plan Nacional de I+D+I" and co-funded by ISCIII-Subdirección General de Evaluacioón and "Fondo Europeo de Desarrollo Regional"(FEDER).D. Domínguez-Villanueva is funded by "Plan Nacional de I+D+I" and co-funded by ISCIII-Subdirección General de Evaluación and "Fondo Europeo de Desarrollo Regional"(FEDER). M. Gomes-Fernandes is funded by CAPES Foundation, Ministry of Education of Brazil (Brasılia, Brazil). Maisem Laabei was supported by a joint ERS/SEPAR fellowship (LTRF2015).This work also received a grant from the Spanish Society of Pneumology and Thoracic Surgery (SEPAR054/2011).Objective. Characterization of Staphylococcus aureus clinical isolates derived from lower respiratory tract infections (LRTIs), and correlation between the functionality of the accessory gene regulator (Agr) and genotypic and phenotypic characteristics, clinical variables and clinical outcome. Methods. S aureus isolates derived from LRTIs and control groups (nasal carriage and bacteraemia) were genotyped using StaphyType DNA microarray. Agr activity was evaluated using the CAMP synergistic haemolysis assay and the Vesicle Lysis Test (VLT). Discordant strains were analysed by quantitative reverse- transcriptase real-time PCR (qRT-PCR). Results. Agr was functional in 79.7% and 84.5% of strains according to the CAMP and VLT assays respectively. Higher concordance with RNAIII expression measured by qRT-PCR was observed with the VLT assay (76.2%) compared with the CAMP assay (23.8%). No statistically significant differences were observed in Agr functionality between the study groups, nor the phenotypical/genotypical bacterial characteristics. No association between increased mortality/respiratory complications and Agr function was observed. Conclusions. Agr activity was high (82.2%) in isolates from LRTIs suggesting the importance of this global regulator in lower respiratory tract colonisation and infection. However, equally high Agr activity was observed in isolates derived from nasal carriage and bacteraemia, contradictory to previous observations. Agr functionality measured by the VLT assay was superior to CAMP assay