Improving the Diagnosis of Bloodstream Infections : PCR Coupled with Mass Spectrometry

The reference method for the diagnosis of bloodstream infections is blood culture followed by biochemical identification and antibiotic susceptibility testing of the isolated pathogen. This process requires 48 to 72 hours. The rapid administration of the most appropriate antimicrobial treatment is crucial for the survival of septic patients; therefore, a rapid method that enables diagnosis directly from analysis of a blood sample without culture is needed. A recently developed platform that couples broad-range PCR amplification of pathogen DNA with electrospray ionization mass spectrometry (PCR/ESI-MS) has the ability to identify virtually any microorganism from direct clinical specimens. To date, two clinical evaluations of the PCR/ESI-MS technology for the diagnosis of bloodstream infections from whole blood have been published. Here we discuss them and describe recent improvements that result in an enhanced sensitivity. Other commercially available assays for the molecular diagnosis of bloodstream infections from whole blood are also reviewed. The use of highly sensitive molecular diagnostic methods in combination with conventional procedures could substantially improve the management of septic patients

Macrolide resistance and molecular typing of Mycoplasma pneumoniae infections during a 4 year period in Spain

Mycoplasma pneumoniae (MP) causes community-acquired pneumonia affecting mainly children, and tends to produce cyclic outbreaks. The widespread use of macrolides is increasing resistance rates to these antibiotics. Molecular tools can help in diagnosis, typing and resistance detection, leading to better patient management. To assess the MP genotypes and resistance pattern circulating in our area while comparing serological and molecular diagnosis of MP. Molecular and serological diagnosis of MP was performed in 821 samples collected in Badalona (Barcelona, Spain) from 2013 to 2017. Multiple locus variable number tandem repeat analysis (MLVA) and macrolide resistance detection by pyrosequencing were performed in those cases positive by PCR. Presence of respiratory viruses and relevant clinical data were also recorded. MP was detected in 16.8% of cases by PCR, with an overall agreement with serology of 76%. Eleven different MLVA types were identified, with 4-5-7-2 (50.1%) and 3-5-6-2 (29.2%) being the most abundant, with the latter showing a seasonal increase during the study. A total of 8% of the strains harboured a point substitution associated with macrolide resistance, corresponding mainly to an A2063G 23S rRNA mutation and directly related to previous macrolide therapy. Analysis of respiratory viruses showed viral coinfections in most cases. Serological and molecular tools combined could improve MP diagnosis and the analysis of its infection patterns. Macrolide resistance is associated with previous therapy. Given that MP pneumonia usually resolves spontaneously, it should be reconsidered whether antibiotic treatment is suitable for all cases

A Simple Polymicrobial Biofilm Keratinocyte Colonization Model for Exploring Interactions Between Commensals, Pathogens and Antimicrobials

© 2020 Jordana-Lluch, Garcia, Kingdon, Singh, Alexander, Williams and Hardie. Skin offers protection against external insults, with the skin microbiota playing a crucial defensive role against pathogens that gain access when the skin barrier is breached. Linkages between skin microbes, biofilms and disease have not been well established although single-species biofilm formation by skin microbiota in vitro has been extensively studied. Consequently, the purpose of this work was to optimize and validate a simple polymicrobial biofilm keratinocyte model for investigating commensal, pathogen and keratinocyte interactions and for evaluating therapeutic agents or health promoting interventions. The model incorporates the commensals (Staphylococcus epidermidis and Micrococcus luteus) and pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) which form robust polymicrobial biofilms on immortalized keratinocytes (HaCat cells). We observed that the commensals reduce the damage caused to the keratinocyte monolayer by either pathogen. When the commensals were combined with P. aeruginosa and S. aureus, much thinner biofilms were observed than those formed by the pathogens alone. When P. aeruginosa was inoculated with S. epidermidis in the presence or absence of M. luteus, the commensals formed a layer between the keratinocytes and pathogen. Although S. aureus completely inhibited the growth of M. luteus in dual-species biofilms, inclusion of S. epidermidis in triple or quadruple species biofilms, enabled M. luteus to retain viability. Using this polymicrobial biofilm keratinocyte model, we demonstrate that a quorum sensing (QS) deficient S. aureus agr mutant, in contrast to the parent, failed to damage the keratinocyte monolayer unless supplied with the exogenous cognate autoinducing peptide. In addition, we show that treatment of the polymicrobial keratinocyte model with nanoparticles containing an inhibitor of the PQS QS system reduced biofilm thickness and P. aeruginosa localization in mono- and polymicrobial biofilms

Dual Bioresponsive Antibiotic and Quorum Sensing Inhibitor Combination Nanoparticles for Treatment of Pseudomonas aeruginosa Biofilms In Vitro and Ex Vivo

Many debilitating infections result from persistent microbial biofilms that do not respond to conventional antibiotic regimens. A potential method to treat such chronic infections is to combine agents which interfere with bacterial biofilm development together with an antibiotic in a single formulation. Here, we explore the use of a new bioresponsive polymer formulation derived from specifically modified alginate nanoparticles (NPs) in order to deliver ciprofloxacin (CIP) in combination with the quorum sensing inhibitor (QSI) 3-amino-7-chloro-2-nonylquinazolin-4(3H)-one (ACNQ) to mature Pseudomonas aeruginosa biofilms. The alginate NPs were engineered to incorporate a pH-responsive linker between the polysaccharide backbone and the QSI, and to encapsulate CIP via charge-charge interactions of the positively-charged drug with the carboxyl residues of the alginate matrix. In this way, a dual-action release of antibiotic and QSI was designed for the low-pH regions of a biofilm, involving cleavage of the QSI-linker to the alginate matrix and reduced charge-charge interactions between CIP and the polysaccharide as the alginate carboxyl side-chains protonated. When tested in a biofilm model the concomitant release of CIP+QSI from the pH-responsive nanoparticles significantly reduced the viability of the biofilm compared with CIP treatment alone. In addition, the alginate NPs were shown to penetrate deeply into P. aeruginosa biofilms, which we attribute in part to the charges of the NPs and the release of the QSI agent. Finally, we tested the formulation in both a 2D keratinocyte and a 3D ex-vivo skin infection model. The dual-action bio-responsive QSI and CIP release nanoparticles effectively cleared the infection in the latter, suggesting considerable promise for combination therapeutics which prevent biofilm formation as well as effectively killing mature P. aeruginosa biofilms

Relevance of Baseline Viral Genetic Heterogeneity and Host Factors for Treatment Outcome Prediction in Hepatitis C Virus 1b-Infected Patients

Only about 50% of patients chronically infected with HCV genotype 1 (HCV-1) respond to treatment with pegylated interferon-alfa and ribavirin (dual therapy), and protease inhibitors have to be administered together with these drugs increasing costs and side-effects. We aimed to develop a predictive model of treatment response based on a combination of baseline clinical and viral parameters. Seventy-four patients chronically infected with HCV-1b and treated with dual therapy were studied (53 retrospectively −training group−, and 21 prospectively −validation group−). Host and viral-related factors (viral load, and genetic variability in the E1-E2, core and Interferon Sensitivity Determining Region) were assessed. Multivariate discriminant analysis and decision tree analysis were used to develop predictive models on the training group, which were then validated in the validation group. A multivariate discriminant predictive model was generated including the following variables in decreasing order of significance: the number of viral variants in the E1-E2 region, an amino acid substitution pattern in the viral core region, the IL28B polymorphism, serum GGT and ALT levels, and viral load. Using this model treatment outcome was accurately predicted in the training group (AUROC = 0.9444; 96.3% specificity, 94.7% PPV, 75% sensitivity, 81% NPV), and the accuracy remained high in the validation group (AUROC = 0.8148, 88.9% specificity, 90.0% PPV, 75.0% sensitivity, 72.7% NPV). A second model was obtained by a decision tree analysis and showed a similarly high accuracy in the training group but a worse reproducibility in the validation group (AUROC = 0.9072 vs. 0.7361, respectively). The baseline predictive models obtained including both host and viral variables had a high positive predictive value in our population of Spanish HCV-1b treatment naïve patients. Accurately identifying those patients that would respond to the dual therapy could help reducing implementation costs and additional side effects of new treatment regimens

The balance between antibiotic resistance and fitness/virulence in Pseudomonas aeruginosa: an update on basic knowledge and fundamental research

The interplay between antibiotic resistance and bacterial fitness/virulence has attracted the interest of researchers for decades because of its therapeutic implications, since it is classically assumed that resistance usually entails certain biological costs. Reviews on this topic revise the published data from a general point of view, including studies based on clinical strains or in vitro-evolved mutants in which the resistance phenotype is seen as a final outcome, i.e., a combination of mechanisms. However, a review analyzing the resistance/fitness balance from the basic research perspective, compiling studies in which the different resistance pathways and respective biological costs are individually approached, was missing. Here we cover this gap, specifically focusing on Pseudomonas aeruginosa, a pathogen that stands out because of its extraordinary capacity for resistance development and for which a considerable number of recent and particular data on the interplay with fitness/virulence have been released. The revised information, split into horizontally-acquired vs. mutation-driven resistance, suggests a great complexity and even controversy in the resistance-fitness/virulence balance in the acute infection context, with results ranging from high costs linked to certain pathways to others that are seemingly cost-free or even cases of resistance mechanisms contributing to increased pathogenic capacities. The elusive mechanistic basis for some enigmatic data, knowledge gaps, and possibilities for therapeutic exploitation are discussed. The information gathered suggests that resistance-fitness/virulence interplay may be a source of potential antipseudomonal targets and thus, this review poses the elementary first step for the future development of these strategies harnessing certain resistance-associated biological burdens

Noves tecnologies per al diagnòstic molecular de la sèpsia

La sèpsia és la resposta sistèmica de l’organisme, en la que es produeixen un conjunt de canvis humorals i cel·lulars, com a resposta a la invasió sanguínia per microorganismes i/o les seves toxines. En l’evolució clínica del pacient intervindran diferents factors com les característiques del microorganisme (inòcul, virulència), la immunocompetència de l’hostatger (patologia de base, factors genètics,...) i la rapidesa en administrar el tractament antibiòtic i de suport adequats. Degut a l’augment de l’expectativa de vida i dels tractaments i manipulacions exploratòries cada vegada més agressius, la incidència de sèpsia s’incrementa any darrere any. Aquesta síndrome té una mortalitat elevada, essent-ne la primera causa a la Unitats de Cures Intensives. A més a més, s’ha demostrat que aquesta s’incrementa en els malats que reben un tractament antibiòtic inadequat. Per tant, la rapidesa en l‘obtenció del diagnòstic microbiològic és crucial per a la disminució de la mortalitat. Actualment, el mètode de referència (gold standard) per al diagnòstic microbiològic de la sèpsia continua sent l’hemocultiu. La seva principal limitació és la mitjana de 15 hores d’incubació per a l’obtenció d’un resultat positiu i després, de 24-48 hores més per a la identificació i l’antibiograma, el que pot implicar un retard en l’aplicació d’una teràpia antibiòtica efectiva. A més a més, en els malalts en que ja s’ha administrat alguna dosi d’antibiòtic prèviament a la inoculació de l’hemocultiu, el rendiment de les tècniques microbiològiques convencionals és baix, el que fa que, en moltes ocasions, no s’arribi al diagnòstic etiològic i, per tant, es desconegui la sensibilitat als antibiòtics del microorganisme causant de la sèpsia. Tot i que fa diversos anys que existeixen assajos moleculars comercialitzats per a aquesta finalitat, només un (SeptiFAST, Roche, Manheim, Alemanya) ha estat extensament avaluat en l’àmbit hospitalari donant resultats variables i encara no ha estat àmpliament implementat. En aquesta tesi doctoral s’ha avaluat la utilitat de la tecnologia PCR/ESI-MS per al diagnòstic molecular de la sèpsia. Aquesta tècnica es basa en una PCR d’ampli espectre seguida d’una espectrometria de masses d’ionització per electrospray i permet la identificació de bacteris i fongs a partir de mostra directa en unes 6 h. Durant l’avaluació de la primera versió (PLEX-ID), es van obtenir resultats prometedors però la sensibilitat de l’assaig era moderada (43,5%) en comparació amb els mètodes convencionals de diagnòstic. Tot i això, aquesta tecnologia va ser capaç de detectar fins a 12 microorganismes clínicament significantius que no van ser aïllats per l’hemocultiu. Degut a la baixa sensibilitat, es va redissenyar la tecnologia. La nova versió, anomenada IRIDICA, presenta com a canvi més important la utilització de 5 mL de sang (en comparació dels 1,250 mL utilitzats amb el PLEX-ID). Amb la nova versió de la tecnologia s’ha obtingut una sensibilitat del 73% en comparació amb els mètodes convencionals. A més a més, IRIDICA va detectar fins a 41 microorganismes clínicament significatius en pacients amb hemocultiu negatiu, la majoria dels quals ja havien rebut tractament antibiòtic. Aquesta tesi també explora la utilitat d’aquesta tècnica en diferents tipus de pacients. Es van analitzar mostres de pacients provinents del Servei d’Urgències i de pacients ingressats a la Unitat de Cures Intensives. En aquest últim grup, IRIDICA va demostrar una millor sensibilitat (83,0%) en comparació als mètodes convencionals. Aquests resultats són concordants amb un estudi multicèntric recentment publicat on s’avalua aquesta tècnica per al diagnòstic molecular de la sèpsia en malats crítics i que inclou pacients diferents Unitats de Cures Intensives de sis països europeus. Per tant, IRIDICA és un sistema robust, ràpid i fiable per a la detecció de microorganismes en sang total i que ha demostrat que incrementa el nombre de casos de sèpsia confirmada microbiològicament incloent els pacients que ja estan en tractament amb antimicrobians. La utilització d’aquest sistema tindria més rellevància en malalts crítics ja que permetria una identificació precoç del microorganisme causant de la sèpsia. Per tant, es podria adequar abans el tractament antibiòtic, millorant la taxa de supervivència i el maneig del pacient.Sepsis is a systemic response of the organism to the invasion by microorganisms and/or their toxins in the blood, characterized by a variety of humoral and cellular changes. Several factors will influencethe clinical outcome of the patient, such as the characteristics of the microorganism (inoculum, virulence), immunocompetence of the host (pathology, genetic factors, etc.) and the time elapsed until the administration of antibiotic treatment and appropriate support. Due to increased life expectancy and more aggressive treatments and exploratory manipulations, the incidence of sepsis increases year after year. This syndrome has a high mortality, being the first cause of death in the Intensive Care Unit. Moreover, it has been shown that the mortality is higher in patients receiving inadequate antibiotic treatment. Therefore, obtaining a rapid microbiological diagnosis is crucial in order to decrease mortality. Currently, the reference method (gold standard) for the microbiological diagnosis of sepsis is blood culture. Its main limitation is the average of 15 hours of incubation needed to obtain a positive result, and another 24-48 hours are needed for the identification and the antimicrobial susceptibility testing, which may cause a delay in the implementation of an effective antibiotic therapy. Furthermore, in patients who are already under antibiotic treatment prior to the inoculation of the blood culture, the performance of conventional microbiological techniques is lower, which means that, in many cases, the etiological diagnosis cannot be obtained and, therefore, the sensitivity to antibiotics of the microorganism causing sepsis is not known. Although there are several molecular assays commercially available, only SeptiFAST (Roche, Mannheim, Germany) has been extensively evaluated in the hospital setting showing heterogeneous results and has not been widely implemented yet. This thesis has evaluated the usefulness of the technology PCR/ESI-MS for the molecular diagnosis of sepsis. This technique is based on a broad spectrum PCR followed by mass spectrometry electrospray ionization and is able to identify bacteria and fungi from direct specimens in about 6 hours. During the evaluation of the first version of this technology (PLEX-ID) promising results were obtained but its sensitivity was moderate (43.5%) in comparison with the conventional diagnostic methods. On the other hand, this technology was able to detect up to 12 clinically significant microorganisms that were not isolated by blood culture. However, because of its suboptimal sensitivity, the technology was refurbished. The new version, named IRIDICA, presents, as a major change, the increase of the volume of blood analyzed up to 5 mL (in comparison with the 1.250 mL used for the PLEX-ID). With the new version, the technology achieved a sensitivity of 73.0% in comparison with conventional methods. Furthermore, IRIDICA detected up to 41 clinically significant microorganisms in patients with negative blood culture, most of whom had previously received antibiotic treatment. This thesis explores the usefulness of this technique in different types of patients. We analyzed samples of patients from the Emergency Room and patients admitted to the Intensive Care Unit. In the latter group, IRIDICA showed a higher sensitivity (83.0%) in comparison with conventional methods. These results are concordant with a recently published multicentric study that evaluated this technique for the molecular diagnosis of sepsis in critically ill patients admitted to the Intensive Care Units from six European countries. In conclusion, IRIDICA is a robust, rapid and reliable technology for the detection of microorganisms directly from whole blood. This technology would increase the number of microbiologically confirmed sepsis cases, even in the presence of antimicrobial treatment. The utilization of this system would provide an early identification of the microorganism causing sepsis, with an improved performance in critically ill patients. Therefore, IRIDICA would lead to a prompter administration of the antimicrobial treatment, thus improving the survival rate and patient management

Improving the Diagnosis of Bloodstream Infections: PCR Coupled with Mass Spectrometry

The reference method for the diagnosis of bloodstream infections is blood culture followed by biochemical identification and antibiotic susceptibility testing of the isolated pathogen. This process requires 48 to 72 hours. The rapid administration of the most appropriate antimicrobial treatment is crucial for the survival of septic patients; therefore, a rapid method that enables diagnosis directly from analysis of a blood sample without culture is needed. A recently developed platform that couples broad-range PCR amplification of pathogen DNA with electrospray ionization mass spectrometry (PCR/ESI-MS) has the ability to identify virtually any microorganism from direct clinical specimens. To date, two clinical evaluations of the PCR/ESI-MS technology for the diagnosis of bloodstream infections from whole blood have been published. Here we discuss them and describe recent improvements that result in an enhanced sensitivity. Other commercially available assays for the molecular diagnosis of bloodstream infections from whole blood are also reviewed. The use of highly sensitive molecular diagnostic methods in combination with conventional procedures could substantially improve the management of septic patients

Noves tecnologies per al diagnòstic molecular de la sèpsia

La sèpsia és la resposta sistèmica de l'organisme, en la que es produeixen un conjunt de canvis humorals i cel·lulars, com a resposta a la invasió sanguínia per microorganismes i/o les seves toxines. En l'evolució clínica del pacient intervindran diferents factors com les característiques del microorganisme (inòcul, virulència), la immunocompetència de l'hostatger (patologia de base, factors genètics,...) i la rapidesa en administrar el tractament antibiòtic i de suport adequats. Degut a l'augment de l'expectativa de vida i dels tractaments i manipulacions exploratòries cada vegada més agressius, la incidència de sèpsia s'incrementa any darrere any. Aquesta síndrome té una mortalitat elevada, essent-ne la primera causa a la Unitats de Cures Intensives. A més a més, s'ha demostrat que aquesta s'incrementa en els malats que reben un tractament antibiòtic inadequat. Per tant, la rapidesa en l'obtenció del diagnòstic microbiològic és crucial per a la disminució de la mortalitat. Actualment, el mètode de referència (gold standard) per al diagnòstic microbiològic de la sèpsia continua sent l'hemocultiu. La seva principal limitació és la mitjana de 15 hores d'incubació per a l'obtenció d'un resultat positiu i després, de 24-48 hores més per a la identificació i l'antibiograma, el que pot implicar un retard en l'aplicació d'una teràpia antibiòtica efectiva. A més a més, en els malalts en que ja s'ha administrat alguna dosi d'antibiòtic prèviament a la inoculació de l'hemocultiu, el rendiment de les tècniques microbiològiques convencionals és baix, el que fa que, en moltes ocasions, no s'arribi al diagnòstic etiològic i, per tant, es desconegui la sensibilitat als antibiòtics del microorganisme causant de la sèpsia. Tot i que fa diversos anys que existeixen assajos moleculars comercialitzats per a aquesta finalitat, només un (SeptiFAST, Roche, Manheim, Alemanya) ha estat extensament avaluat en l'àmbit hospitalari donant resultats variables i encara no ha estat àmpliament implementat. En aquesta tesi doctoral s'ha avaluat la utilitat de la tecnologia PCR/ESI-MS per al diagnòstic molecular de la sèpsia. Aquesta tècnica es basa en una PCR d'ampli espectre seguida d'una espectrometria de masses d'ionització per electrospray i permet la identificació de bacteris i fongs a partir de mostra directa en unes 6 h. Durant l'avaluació de la primera versió (PLEX-ID), es van obtenir resultats prometedors però la sensibilitat de l'assaig era moderada (43,5%) en comparació amb els mètodes convencionals de diagnòstic. Tot i això, aquesta tecnologia va ser capaç de detectar fins a 12 microorganismes clínicament significantius que no van ser aïllats per l'hemocultiu. Degut a la baixa sensibilitat, es va redissenyar la tecnologia. La nova versió, anomenada IRIDICA, presenta com a canvi més important la utilització de 5 mL de sang (en comparació dels 1,250 mL utilitzats amb el PLEX-ID). Amb la nova versió de la tecnologia s'ha obtingut una sensibilitat del 73% en comparació amb els mètodes convencionals. A més a més, IRIDICA va detectar fins a 41 microorganismes clínicament significatius en pacients amb hemocultiu negatiu, la majoria dels quals ja havien rebut tractament antibiòtic. Aquesta tesi també explora la utilitat d'aquesta tècnica en diferents tipus de pacients. Es van analitzar mostres de pacients provinents del Servei d'Urgències i de pacients ingressats a la Unitat de Cures Intensives. En aquest últim grup, IRIDICA va demostrar una millor sensibilitat (83,0%) en comparació als mètodes convencionals. Aquests resultats són concordants amb un estudi multicèntric recentment publicat on s'avalua aquesta tècnica per al diagnòstic molecular de la sèpsia en malats crítics i que inclou pacients diferents Unitats de Cures Intensives de sis països europeus. Per tant, IRIDICA és un sistema robust, ràpid i fiable per a la detecció de microorganismes en sang total i que ha demostrat que incrementa el nombre de casos de sèpsia confirmada microbiològicament incloent els pacients que ja estan en tractament amb antimicrobians. La utilització d'aquest sistema tindria més rellevància en malalts crítics ja que permetria una identificació precoç del microorganisme causant de la sèpsia. Per tant, es podria adequar abans el tractament antibiòtic, millorant la taxa de supervivència i el maneig del pacient.Sepsis is a systemic response of the organism to the invasion by microorganisms and/or their toxins in the blood, characterized by a variety of humoral and cellular changes. Several factors will influencethe clinical outcome of the patient, such as the characteristics of the microorganism (inoculum, virulence), immunocompetence of the host (pathology, genetic factors, etc.) and the time elapsed until the administration of antibiotic treatment and appropriate support. Due to increased life expectancy and more aggressive treatments and exploratory manipulations, the incidence of sepsis increases year after year. This syndrome has a high mortality, being the first cause of death in the Intensive Care Unit. Moreover, it has been shown that the mortality is higher in patients receiving inadequate antibiotic treatment. Therefore, obtaining a rapid microbiological diagnosis is crucial in order to decrease mortality. Currently, the reference method (gold standard) for the microbiological diagnosis of sepsis is blood culture. Its main limitation is the average of 15 hours of incubation needed to obtain a positive result, and another 24-48 hours are needed for the identification and the antimicrobial susceptibility testing, which may cause a delay in the implementation of an effective antibiotic therapy. Furthermore, in patients who are already under antibiotic treatment prior to the inoculation of the blood culture, the performance of conventional microbiological techniques is lower, which means that, in many cases, the etiological diagnosis cannot be obtained and, therefore, the sensitivity to antibiotics of the microorganism causing sepsis is not known. Although there are several molecular assays commercially available, only SeptiFAST (Roche, Mannheim, Germany) has been extensively evaluated in the hospital setting showing heterogeneous results and has not been widely implemented yet. This thesis has evaluated the usefulness of the technology PCR/ESI-MS for the molecular diagnosis of sepsis. This technique is based on a broad spectrum PCR followed by mass spectrometry electrospray ionization and is able to identify bacteria and fungi from direct specimens in about 6 hours. During the evaluation of the first version of this technology (PLEX-ID) promising results were obtained but its sensitivity was moderate (43.5%) in comparison with the conventional diagnostic methods. On the other hand, this technology was able to detect up to 12 clinically significant microorganisms that were not isolated by blood culture. However, because of its suboptimal sensitivity, the technology was refurbished. The new version, named IRIDICA, presents, as a major change, the increase of the volume of blood analyzed up to 5 mL (in comparison with the 1.250 mL used for the PLEX-ID). With the new version, the technology achieved a sensitivity of 73.0% in comparison with conventional methods. Furthermore, IRIDICA detected up to 41 clinically significant microorganisms in patients with negative blood culture, most of whom had previously received antibiotic treatment. This thesis explores the usefulness of this technique in different types of patients. We analyzed samples of patients from the Emergency Room and patients admitted to the Intensive Care Unit. In the latter group, IRIDICA showed a higher sensitivity (83.0%) in comparison with conventional methods. These results are concordant with a recently published multicentric study that evaluated this technique for the molecular diagnosis of sepsis in critically ill patients admitted to the Intensive Care Units from six European countries. In conclusion, IRIDICA is a robust, rapid and reliable technology for the detection of microorganisms directly from whole blood. This technology would increase the number of microbiologically confirmed sepsis cases, even in the presence of antimicrobial treatment. The utilization of this system would provide an early identification of the microorganism causing sepsis, with an improved performance in critically ill patients. Therefore, IRIDICA would lead to a prompter administration of the antimicrobial treatment, thus improving the survival rate and patient management