Centro Nacional de Microbiología (CNM)

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12105/19609

El Centro Nacional de Microbiología (CNM) es uno de los centros pertenecientes al Instituto de Salud Carlos III que proporciona apoyo científico-técnico a la Administración General del Estado, a las Comunidades Autónomas y al Sistema Nacional de Salud (SNS), tal y como se recoge en La Ley General de Sanidad (Ley 14/1986, de 25 de abril) y el Estatuto del Instituto de Salud Carlos III (RD 375/2001, de 6 abril y su posterior reforma, RD 1672/2009, de 6 de noviembre). La función específica del CNM es el control de las enfermedades infecciosas para lo que ofrece servicios de diagnóstico y referencia, manteniendo además programas de investigación, tanto básica como orientada, relacionados con la prevención, el diagnóstico y el tratamiento de estas enfermedades. Dispone de un sistema de gestión de calidad conforme a la norma UNE-EN ISO 9001, certificado por AENOR, para la recepción de muestras biológicas, así como varias técnicas y servicios acreditados por ENAC según la norma UNE-EN ISO 15189.

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High prevalence of azole resistance among environmental isolates from outdoor air in Madrid, Spain.
(Frontiers Media, 2026-01-23) Soto-Debrán, Juan Carlos; Sanchez Iñigo, Francisco Javier; Calvo-López, Alejandro B; Alguacil-Cuéllar, Laura; Hrynzovska, Anastasiia A; Mellado, Emilia; Garcia Dos Santos-Alves, Saul; Alastruey-Izquierdo, Ana; Instituto de Salud Carlos III
Introduction: Aspergillus fumigatus has been designated by the World Health Organization (WHO) as a critical fungal pathogen. Its spores are commonly present in the air and are inhaled daily. Azoles are the first-line treatment for Aspergillus infections, but the emergence of resistance is a growing concern. However, limited data exist on the occurrence of azole-resistant A. fumigatus in the outdoor environment in Spain. Methods: This study aimed to investigate the prevalence of azole-resistant A. fumigatus isolates in outdoor air at two distinct locations in Madrid. We characterized the isolates using TRESPERG genotyping and examined the underlying molecular mechanisms responsible for azole resistance development. Results: Azole-resistant A. fumigatus isolates were found in 55% of the 20 air samples collected. Among the 200 A. fumigatus isolates analyzed, 38.5% were azole resistant and were classified into 10 different genotypes. Notably, the TR34/L98H mutation in Cyp51A was found in 77% of the resistant isolates, while 23% showed no mutations in the screened targets (cyp51A, cyp51B, or hmg1). Discussion: This study revealed a high prevalence of azole-resistant A. fumigatus in outdoor environmental air, with the TR34/L98H mutation being the main mechanism of azole resistance. A close genetic relationship was observed among the resistant isolates. This research underscores the need for continued monitoring of environmental azole-resistant A. fumigatus isolates and highlights the importance of understanding genetic diversity and resistance mechanisms to develop effective strategies for fungal infection control.
Elevated Zonulin-1 is Associated With an Increased Risk of Non-AIDS-Defining Cancers in People With HIV With Suboptimal Immune Recovery: A Case-Cohort Study.
(Wiley, 2026-04) Pita-Martínez, Carlos; Muñoz-García, Paula; Martinez, Isidoro; Rava, Marta; Bautista Hernández, Azucena; Pérez-Somarriba, Juncal; Campins, Antoni; Novella Mena, María; Vera, Francisco; Montero-Alonso, Marta; Jimenez-Sousa, Maria Angeles; Resino, Salvador; Martín-Escolano, Rubén; CoRIS cohort; Instituto de Salud Carlos III; Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Agencia Estatal de Investigación (España); Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas); Comunidad de Madrid (España); Centro de Investigación Biomédica en Red - CIBERBBN (Bioingeniería, Biomateriales y Nanomedicina)
People with HIV (PWH) on antiretroviral therapy (ART) exhibit a heightened risk of non-AIDS-defining cancers (NADCs), linked to chronic inflammation driven by microbial translocation. We investigated whether plasma biomarkers of gut barrier dysfunction are associated with NADC risk, hypothesizing this association is modified by immune status. We conducted a case-cohort study nested within the Spanish CoRIS cohort. Plasma levels of zonulin-1, lipopolysaccharide, LPS-binding protein, and flagellin were measured in 71 incident NADC cases and a 261-individuals subcohort. Multivariable Cox proportional hazards models were used to estimate NADC risk, testing for effect modification by baseline CD4 + T-cell count. Zonulin-1 was not independently associated with NADC risk. However, a strong statistical interaction with baseline CD4 + T-cell count was detected (p = 0.001). Among individuals with suboptimal immune recovery (defined as CD4 + T-cell count < 500 cells/mm³), higher zonulin-1 levels were associated with a nearly threefold increased NADC risk (aHR = 2.94 (1.28-6.76)). Conversely, no association was observed in those with robust immune reconstitution (≥ 500 cells/mm³; aHR= 0.84 (0.41-1.72)). Other biomarkers showed no association. This supports a "two-hit" model of carcinogenesis where a compromised gut barrier and impaired immune competence are associated with an increased risk of cancer. Zonulin-1 is a key biomarker for identifying this high-risk phenotype, suggesting targeted cancer prevention strategies restoring gut integrity.
Lipid and immune dysregulation and risk of metabolic disorders after HCV clearance in HIV/HCV-coinfected participants with cACLD: a retrospective study.
(Frontiers Media, 2026-01-12) Virseda-Berdices, Ana; Requena, Belen; Berenguer, Juan; González-García, Juan; Gonzalez-Riano, Carolina; Díez, Cristina; Hontañon, Victor; Fernandez-Rodriguez, Amanda; Barbas, Coral; Resino, Salvador; Martín-Escolano, Rubén; Jimenez-Sousa, Maria Angeles; Instituto de Salud Carlos III; Agencia Estatal de Investigación (España); Unión Europea. Comisión Europea. NextGenerationEU; Comunidad de Madrid (España)
Introduction: People with HIV and chronic hepatitis C may develop metabolic complications after sustained virologic response (SVR), possibly due to persistent molecular alterations induced by HCV. This study aimed to identify baseline (pre-treatment) lipid and immune biomarkers associated with post-SVR metabolic events in HIV/HCV-coinfected participants with compensated advanced chronic liver disease (cACLD) receiving long-term suppressive antiretroviral therapy. Methods: We conducted a retrospective study of 56 HIV/HCV-coinfected participants with cACLD. Untargeted lipidomic profiling was performed on baseline plasma samples using a liquid-chromatography-mass spectrometer. The outcome was the development of metabolic events (diabetes mellitus and/or hyperlipidemia) during follow-up, up to seven years post-HCV treatment. Statistical analyses included orthogonal partial least squares discriminant analysis (OPLS-DA), Cox regressions models, and Spearman correlations with inflammation-related biomarkers and immune checkpoint proteins, with multiple comparison corrections using the false discovery rate. Results: 25% participants developed metabolic events after SVR. OPLS-DA identified 163 lipid species (VIP scores≥1) associated with these events, and adjusted Cox regression confirmed significant associations for 24 of them. Lysophosphatidylcholines (LPCs) were the most prevalent, with higher baseline levels linked to increased metabolic risk. Participants who developed events also had higher levels of plasmalogens LPC (O-LPC), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), lysophosphatidic acid (LPA), and phosphosphatidylcholine (PC). Several lipid species correlated positively with the pro-inflammatory cytokine IL-18, the anti-inflammatory suppressor IL-1RA, and the immune checkpoint proteins IDO and S100A8/A9. Discussion: Pre-treatment lipid and immune dysregulation was associated with post-SVR metabolic events in HIV/HCV-coinfected participants, suggesting that HCV may leave a lasting metabolic imprint that contributes to adverse outcomes after viral clearance.
HIV status defines distinct immunological drivers of persistent portal hypertension after HCV cure in people with advanced cirrhosis.
(Frontiers Media, 2026-02-02) Martín-Escolano, Rubén; Fernandez-Rodriguez, Amanda; Tarancon-Diez, Laura; Berenguer, Juan; Codina Márquez, Helena; Amigot-Sánchez, Rafael; González-García, Juan; Hontañón, Víctor; Pérez-Latorre, Leire; Ibañez-Samaniego, Luis; Llop-Herrera, Elba; Olveira, Antonio; Díaz, Laura; Martinez, Isidoro; Jimenez-Sousa, Maria Angeles; Resino, Salvador; Instituto de Salud Carlos III; Agencia Estatal de Investigación (España); Unión Europea. Comisión Europea. NextGenerationEU; Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas); Comunidad de Madrid (España)
Introduction: The immunological drivers of portal hypertension regression after hepatitis C virus (HCV) cure are poorly understood, particularly in the context of human immunodeficiency virus (HIV) coinfection We aimed to identify baseline immune signatures predicting the evolution of the hepatic venous pressure gradient (HVPG) in people with and without HIV (PWH/PWoH). Methods: We prospectively followed 41 individuals with advanced cirrhosis (18 PWoH, 23 PWH) who were cured of HCV with direct-acting antivirals (DAA). Baseline plasma and cellular immune markers were extensively profiled using multiplex assays and flow cytometry. We used mixed-effects modeling to test for associations between these baseline immune features and the change in HVPG over a 48-week follow-up period, with q-values controlling for false discoveries. Results: Two distinct immunological profiles of impaired HVPG regression emerged. In PWoH, impaired regression was linked to a broad proinflammatory profile [TNF-α (AMR = 1.13; q=0.012), IL17A (AMR = 1.28; q=0.012), and IL10 (AMR = 1.2; q=0.028)], a widespread total CD4+ T-cell activation [HLA-DR+ (AMR = 1.44; q<0.001) and CD38+HLA-DR+ (AMR = 1.3; q=0.007)], and robust activation across central memory (CM) and effector memory (EM) subsets. Conversely, in PWH, impaired HVPG regression was associated with sVCAM-1 (AMR = 1.58; q=0.096), and a more focused activation within EM (HLA-DR+, AMR = 1.08; q=0.030) and TemRA (CD38+HLA-DR+, AMR = 1.12; q=0.030) CD4+ T-cells. Discussion: HIV coinfection fundamentally reshapes the immunological landscape of post-cure portal hypertension recovery. The shift from systemic inflammation in PWoH to endothelial dysfunction and T-cell exhaustion in PWH reveals distinct pathological pathways. Understanding these signatures is a crucial step toward developing targeted therapies to promote complete hepatic recovery.
Long-term effects of HCV eradication on lipid profiles associated with MASLD among people with HIV with advanced fibrosis or cirrhosis.
(Elsevier, 2025-12) Virseda-Berdices, Ana; Requena, Belen; Berenguer, Juan; Gónzalez-García, Juan; Gonzalez-Riano, Carolina; Díez, Cristina; Hontañón, Victor; Muñoz-García, Paula; Fernandez-Rodriguez, Amanda; Barbas, Coral; Resino, Salvador; Martín-Escolano, Rubén; Jimenez-Sousa, Maria Angeles; the Marathon Study Group; Instituto de Salud Carlos III; Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas); Unión Europea. Comisión Europea. NextGenerationEU; Comunidad de Madrid (España)
Background: Despite successful hepatitis C virus (HCV) clearance, some individuals continue to experience liver disease progression. Metabolic dysfunction-associated steatotic liver disease (MASLD) may play a key role in this ongoing progression. This study aims to characterize the lipidomic profiles associated with MASLD in individuals coinfected with human immunodeficiency virus (HIV) and HCV with advanced fibrosis or cirrhosis after sustained virologic response (SVR). Methods: We conducted cross-sectional studies in fifty-two HIV/HCV-coinfected individuals. Untargeted lipidomics was performed on plasma samples collected at 1 year and 6 years post-SVR using liquid chromatography-mass spectrometry. The primary outcome was MASLD. Statistical analyses included orthogonal partial least squares discriminant analysis (OPLS-DA) and generalized linear models (GLM), with corrections for multiple comparisons. Results: The prevalence of MASLD was 28.9 % one year after SVR, increasing to 44.8 % six years after SVR. OPLS-DA models identified 225 lipids at 1 year and 167 at 6 years, with a VIP score ≥ 1, distinguishing individuals based on MASLD status. Adjusted GLMs confirmed significant associations between MASLD and 116 lipids at 1 year and 49 at 6 years. At 1 year, most significant lipids were glycerophospholipids (GP), with increased phosphatidylcholines (PC) and phosphatidylethanolamines (PE), and decreased lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE). By 6 years, LPC was the most abundant differential lipid, while triglycerides increased significantly. Conclusions: MASLD was common during follow-up, with changes in lipidomic profiles over time suggesting ongoing metabolic disturbances that may contribute to liver disease progression despite SVR. These findings highlight the need for long-term metabolic and liver health monitoring after HCV eradication in these individuals.
Immune and senescence profiles associated with non-AIDS-defining cancer risk in people with HIV: a case-cohort study.
(Frontiers Media, 2025-10-21) Pita-Martínez, Carlos; Jimenez-Sousa, Maria Angeles; Martinez-Picado, Javier; Rodríguez, Carmen Elena Gómez; Fariñas, Carmen; Galindo, Pepa; Roca-Oporto, Cristina; Santos, Jesús; Muñoz-García, Paula; Rava, Marta; Resino, Salvador; Martín-Escolano, Rubén; Instituto de Salud Carlos III; Agencia Estatal de Investigación (España); Unión Europea. Comisión Europea. NextGenerationEU; Comunidad de Madrid (España); Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas)
Introduction: People with HIV (PWH) on effective antiretroviral therapy (ART) have an increased risk of developing Non-AIDS Defining Cancers (NADCs) compared to the general population, partly due to chronic inflammation and immune dysregulation. This study aimed to identify plasma biomarkers associated with the risk of developing NADCs in a cohort of PWH on ART. Methods: A case-cohort study was conducted within the Spanish CoRIS cohort, including 316 PWH on ART (71 cases and 245-individuals subcohort). Plasma levels of 24 immune regulation and senescence-associated secretory phenotype (SASP) biomarkers were quantified using Luminex technology. Cox proportional hazards regression models with Borgan II weights were used to assess the association between biomarker levels and the risk of NADC development (hazard ratios), adjusting for confounders. Effect modification by gender was also evaluated. Results: Higher baseline plasma levels of twelve biomarkers were significantly associated with increased NADC risk. The strongest associations were found for PD-L2 (aHR=3.33), PAI-1 (aHR=2.27), and MMP-1 (aHR=2.32). However, a distinct, gender-specific pattern was observed, with significant interactions found for nine biomarkers. Most interactions indicated a higher NADC risk increase in females, with the exception of CD80, TNF-β and IP-10, which indicated a relatively lower risk in females compared to males. Discussion: Plasma biomarkers of immune regulation and SASP are associated with NADC risk in PWH on long-term ART, highlighting the importance of gender-specific pathways in NADC development among PWH. Understanding these distinct profiles may guide future strategies for risk stratification, early detection, and personalized preventive care.
Low Specific T-Cell Immunity Against Mpox Elicited in People With HIV-1 and PrEP Users After Subcutaneous Vaccination Compared to Natural Infection.
(Wiley, 2025-07) Calle-Jiménez, Olivia de la; Casado-Fernández, Guiomar; Armenteros-Yeguas, Inés; Lemus-Aguilar, Luis; Baza, Begoña; Pérez-García, Jorge Alfredo; Rodríguez-Añover, Javier; Mateos, Elena; Homen, Reynaldo; Orviz-García, Eva; Cabello, Noemí; Negredo, Anabel; Sánchez-Seco, María Paz; Del Romero, Jorge; Estrada, Vicente; Torres, Montserrat; Coiras, Mayte; National Institutes of Health (Estados Unidos); Ministerio de Ciencia e Innovación (España); Agencia Estatal de Investigación (España); Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Instituto de Salud Carlos III; Centro de Investigación Biomédica en Red - CIBERINFEC (Enfermedades Infecciosas); Comunidad de Madrid (España)
In August 2024, the World Health Organization declared mpox infection a Public Health Emergency of International Concern for second time since 2022 and recommended vaccinating all people at-risk, such as people with HIV-1 (PWH) and prophylaxis pre-exposition (PrEP) users. We recruited PWH and PrEP users who received one or two doses of subcutaneous Imvanex® or Jynneos® and compared specific T-cell immunity with participants who passed mpox natural infection or were Nonexposed to mpox. CD4 + T cells from people who had mpox showed the highest capacity to produce IL-2 (1.8-fold, p = 0.0328), as well as IFNγ (2.5-fold, p = 0.0247), and IL-4 (1.8-fold, p = 0.0373) from naïve CD4 + T cells, in response to MHC-II-restricted mpox-related peptides, compared to vaccinated participants. CD8 + T cells from individuals who had mpox also showed the highest capacity to produce IFN (1.6-fold, p = 0.0321) and TNF (2.1-fold, p = 0.0084) against MHC-I-restricted peptides. Therefore, the most potent and robust T-cell responses were developed after mpox infection, while they were barely detectable after vaccination. These results support the need to explore booster doses or improved vaccines to enhance cellular immunity in at-risk populations. More studies are needed to evaluate the capacity of mpox vaccines to confer long-term protection.
Frequent carriage of resistance mechanisms to β-lactams and biofilm formation in Haemophilus influenzae causing treatment failure and recurrent otitis media in young children.
(https://doi.org/10.1093/jac/dku158, 2014-09) García-Cobos, Silvia; Moscoso, Miriam; Pumarola, Félix; Arroyo, Margarita; Lara Fuella, Noelia; Perez-Vazquez, Maria; Aracil, Belen; Oteo-Iglesias, Jesus; García, Ernesto; Campos, Jose
Objectives: Non-typeable Haemophilus influenzae are a major cause of acute otitis media (AOM), including chronic and recurrent otitis in young children. The objective of this study was to determine whether non-typeable H. influenzae isolates causing these infections produce biofilms and carry resistance mechanisms to β-lactams. Methods: A collection of 48 H. influenzae isolates was obtained by tympanocentesis or from otorrhoea samples from individual patients <3 years of age and diagnosed with recurrent or treatment failure AOM. Each isolate was surveyed for the presence of blaTEM genes, amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3) and biofilm formation in microtitre plates. Results: In 43 of the 48 isolates (89.6%), at least one of the three tested conditions was identified: biofilm formation (83.3%) and resistance mechanisms to β-lactams (33.3%), modifications in the transpeptidase domain of PBP3 being the most prevalent (22.9%), followed by β-lactamase production (10.4%). Additionally, 13 (27.1%) isolates had two or more of these three traits. In relation to biofilm formation, those isolates with an amoxicillin MIC ≤ 0.5 mg/L had higher optical density values than isolates with an amoxicillin MIC ≥ 1 mg/L (Mann-Whitney U-test, P=0.048). Conclusions: These findings suggest that the successful treatment of non-typeable H. influenzae causing chronic and recurrent AOM in young children may be compromised by the high biofilm-forming capacity of the isolates and the presence of β-lactam resistance mechanisms, particularly PBP3 mutations.
Isolates of β-lactamase-negative ampicillin-resistant Haemophilus influenzae causing invasive infections in Spain remain susceptible to cefotaxime and imipenem.
(Oxford University Press, 2014-01) García-Cobos, Silvia; Arroyo, Margarita; Pérez-Vázquez, María; Aracil, Belen; Lara Fuella, Noelia; Oteo-Iglesias, Jesus; Cercenado, Emilia; Campos, Jose
Objectives: The epidemiology of invasive Haemophilus influenzae has changed in recent years. β-Lactamase-negative ampicillin-resistant (BLNAR) invasive isolates have recently been described in Europe but their clinical significance is unclear. Our main goal was to determine whether invasive H. influenzae remains susceptible to β-lactam antibiotics indicated in the treatment of invasive infections. Methods: The antibiotic susceptibility of 307 invasive H. influenzae isolates to seven β-lactam antibiotics was determined by microdilution and interpreted by EUCAST and CLSI breakpoints. We also identified the bla genes, the amino acid substitutions in the transpeptidase domain of penicillin-binding protein 3 (PBP3), the molecular epidemiology of invasive BLNAR isolates by PFGE and MLST, and the time-kill curves of two isolates with PBP3 mutations conferring reduced susceptibility to aminopenicillins and cephalosporins. Results: Of the invasive isolates, 86.6% were non-typeable and 62% were isolated from adults. Decreased susceptibility to β-lactams was due to the BLNAR genotype (gBLNAR; 19.2%) and to β-lactamase production (16.9%). Susceptibility rates to amoxicillin/clavulanic acid, cefotaxime, cefixime and imipenem were greater than 98%. Of 18 gBLNAR non-typeable isolates studied by MLST, 15 different STs were obtained. Amoxicillin and cefotaxime were bactericidal after 2 and 4 h of incubation, respectively. Conclusions: Invasive H. influenzae disease was mainly due to non-typeable isolates infecting adults, and the most common mechanism of β-lactam resistance was mutations in the transpeptidase domain of PBP3. The gBLNAR non-typeable isolates were genetically diverse. The majority of invasive H. influenzae remained susceptible to third-generation cephalosporins; amoxicillin and cefotaxime were bactericidal in two gBLNAR isolates.
The hare (Lepus granatensis) as potential sylvatic reservoir of Leishmania infantum in Spain.
(Elsevier, 2012-11-23) Molina, Ricardo; Jimenez, Isabel; Cruz, Israel; Iriso, A; Martín-Martín, I; Sevillano, O; Melero, S; Bernal, J
Xenodiagnosis of Leishmania infection in hares (Lepus granatensis) from a focus of human leishmaniasis in Fuenlabrada at southwestern Madrid region (Spain) proved that they are infective to Phlebotomus perniciosus. Molecular characterization of isolates obtained from sand flies infected after xenodiagnosis demonstrates that hares were infected by Leishmania infantum. This is the first evidence of the transmission of L. infantum from hares to sand flies. Moreover the results confirm the role that these animals can play as wild reservoirs of leishmaniasis for the recent outbreak of visceral leishmaniasis in Madrid.
Key role of whole genome sequencing in resolving an international outbreak of monophasic Salmonella Typhimurium linked to chocolate products.
(BioMed Central (BMC), 2025-02-20) Laisnez, Valeska; Vusirikala, Amoolya; Nielsen, Charlotte Salgaard; Cantaert, Vera; Delbrassinne, Laurence; Mattheus, Wesley; Verhaegen, Bavo; Delamare, Hugues; Jourdan-Da Silva, Nathalie; Lachmann, Raskit; Simon, Sandra; Cormican, Martin; Garvey, Patricia; McKeown, Paul; Stephan, Roger; Brown, Derek; Browning, Lynda; Hoban, Ann; Larkin, Lesley; Pardos de la Gandara, Maria; Jernberg, Cecilia; Takkinen, Johanna; Balasegaram, Sooria; Van Cauteren, Dieter; Varela Martinez, Maria del Carmen; Herrera-León, Silvia; S. Typhimurium Outbreak Investigation Group; Varela Martinez, Maria del Carmen, col.; Herrera-León, Silvia, col.
Background: In February 2022, the United Kingdom (UK) detected a cluster of monophasic Salmonella Typhimurium based on whole genome sequencing (WGS). Subsequently, several countries reported cases belonging to this cluster. Epidemiological, microbiological and traceability investigations pointed toward a chocolate food business operator (FBO) in Belgium. We describe the magnitude of the outbreak, investigations performed and control measures taken. Methods: Cases were ascertained based on internationally agreed case definitions and interviewed about food consumption prior to disease onset. Analytical epidemiological studies were conducted by the UK and Ireland. The Belgian food safety authority (FSA) coordinated microbiological and traceability investigations. Results: A total of 456 cases (61% female), belonging to two genetically different WGS clusters, in 14 countries of the European Union, the UK, Switzerland, Canada and the United States were linked to the outbreak, between December 2021 and June 2022. 87% of cases were younger than 10 years. Brand A chocolate eggs, marketed for children, were reported as consumed by 168 cases (80%) with information. Analytical studies in the UK and Ireland showed a significantly higher odds of disease associated with consumption of brand A chocolate products. Retrospective investigations by the FSA revealed that routine samples (raw materials, intermediate, semi-finished and finished products as well as environmental and rinse oil samples) taken by the FBO linked to the Brand A products between December 2021 and January 2022 had tested positive for salmonella. Nine isolates were submitted to EnteroBase and matched with human isolates from both WGS clusters. The authorization for production was temporarily withdrawn on 8 April 2022 and all products of brand A were recalled worldwide, followed by a decrease in cases. Conclusions: A multi-country salmonellosis outbreak linked to chocolate occurred in the months before Easter 2022. International collaboration between stakeholders from epidemiological, microbiological and food safety entities with rapid sharing of WGS results from human and nonhuman isolates were key in containing the outbreak. Implementation of routine WGS on human and nonhuman isolates will strengthen public health responses to future outbreaks. The magnitude of the outbreak underlines the importance of timely and open communication of FBOs to FSAs in case of salmonella detection.
Viruses Previously Classified as CRF146_BC, a Circulating Recombinant Form of HIV-1 Recently Reported in Brazil, Represent Different Recombinant Forms, One of Which Is Circulating in Spain.
(Multidisciplinary Digital Publishing Institute (MDPI), 2026-01-12) Donoso, Ana; Moreno-Lorenzo, Maria; Delgado, Elena; Cañada-Garcia, Javier Enrique; Iribarren, José Antonio; Salicio, Yolanda; Benito, Sonia; Lorente-Sorolla, Clara; Del Romero-Guerrero, Jorge; Baza-Caraciolo, María Begoña; Díez-Fuertes, Francisco; Zamarrón, Pilar; Téllez, Raquel; Miqueleiz, Ana; Gómez-González, Carmen; Cortizo, Sandra; Morano, Luis; Thomson, Michael M
Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Previous studies have reported the propagation of diverse HIV-1 CRFs of South American origin in Europe. Here, through protease-reverse transcriptase (Pr-RT) and integrase sequence analyses, we identify a Spanish cluster (BC3) branching with a Brazilian virus (10BR_RJ009) previously classified as CRF146_BC. In Pr-RT, BC3 comprised 14 viruses and was nested within a larger cluster, comprising 22 Brazilian viruses and 1 Spanish virus branching outside of BC3. Near full-length genome analyses of five BC3 viruses revealed mosaic structures identical to 10BR_RJ009, with two breakpoints delimiting a ~0.3 kb subtype B fragment within an otherwise subtype C genome. Two other Brazilian viruses previously classified as CRF146_BC (10BR_RJ039 and 01_BR_RGS69) had one and two additional short subtype B fragments, respectively, and failed to group with the 10BR_RJ009/BC3 cluster in subtype C fragments. Based on these results, we contend that 10BR_RJ009 and BC3 viruses, but not 10BR_RJ039 and 01_BR_RGS69, should be classified as CRF146_BC. Bayesian analyses estimated the CRF146_BC emergence in Brazil to be around 1999 and its introduction in Europe around 2011. CRF146_BC is the 10th CRF of South American origin reported to circulate in Europe, reflecting the relationship between South American and European HIV-1 epidemics.
Discontinuous EBOV RNA synthesis events in patients with Ebola virus disease and their relationship to viral load and outcome of infection.
(American Society for Microbiology (ASM), 2025-12-23) Dong, Xiaofeng; Garcia-Dorival, Isabel; Rickett, Natasha Y; Bosworth, Andrew; Smither, Sophie; Dowall, Stuart; Carroll, Miles W; Matthews, David A; Laws, Thomas R; Digard, Paul; Hiscox, Julian A
Ebola virus (EBOV) is a negative strand RNA virus that causes Ebola virus disease (EVD) with a high case fatality rate. During the acute phase of infection, a subsequent fatal outcome is characterized by an increased upregulation of interferon and inflammatory pathways compared to survivors. Replication of genomes from negative strand RNA viruses (and RNA viruses in general) can result in the generation of defective genomes that may interfere with viral replication and stimulation of the innate immune response. We characterized the presence of defective genomes in blood samples from humans who were positive for EBOV and processed by the European Mobile Laboratory during the 2013-2016 West African EVD outbreak. A bioinformatics tool, DI-tector, was used to identify sequence motifs associated with the four different types of defective genomes. The analysis indicated that sequence features indicative of defective genomes were present in blood samples in patients during the acute phase of infection. The most common type of defective genome identified was insertion followed by deletion, 5' copy back and then 3' copy back. In general, the abundance of defective genomes correlated with viral load, but particularly with patient outcome. We postulate that the presence of defective genomes correlates with an upregulation in the interferon response and resultant inflammation and may, therefore, be an important contributory factor in patients with severe EVD.IMPORTANCEEBOV and filoviruses in general are high consequence infectious diseases whose outbreaks can severely impact the lives of those affected. In this study, we show that during EBOV replication in humans, defective genomes can be produced, which complements previous studies in nonhuman primate models of disease and in cell culture. The abundance of these defective genomes correlates with disease outcome in acutely ill patients. In people who go on to die from EVD, they appear to have higher levels of defective genomes than in people who go on to survive infection. This may, in turn, cause a greater upregulation of interferon and inflammation, which are some of the biggest factors in determining disease severity and adverse patient outcome. Therefore, we caution the potential use of defective genomes as a therapy for EVD, as has been proposed for other negative strand RNA viruses.
PPARγ2 Pro12Ala polymorphism is associated with sustained virological response in HIV/HCV-coinfected patients under HCV therapy.
(Wolters Kluwer, 2014-10-01) Fernandez-Rodriguez, Amanda; Berenguer, Juan; Rallón, Norma; Jimenez-Sousa, Maria Angeles; López, Juan Carlos; Soriano, Vicente; Garcia-Alvarez, Monica; Cosín, Jaime; Martínez, Paula; Guzman-Fulgencio, Maria; Miralles, Pilar; Miguel Benito, José; Resino, Salvador
Objectives: To analyze whether peroxisome proliferator-activated receptor gamma (PPARγ2) rs1801282 (Pro12Ala) polymorphism is associated with the response to pegylated-interferon-alpha plus ribavirin treatment in HIV/hepatitis C virus (HCV)-coinfected patients, and whether it is able to predict the outcome of HCV treatment. Design: Retrospective follow-up study. Methods: Two hundred eighty-five naive patients, who started HCV-treatment, were genotyped for PPARγ2 and interleukin 28B polymorphisms. Genetic data were analyzed under dominant inheritance model. Sustained virological response (SVR) was defined as undetectable HCV viremia through 24 weeks after the end of HCV treatment. Results: The variables significantly associated with SVR in a multivariate analysis were HCV-genotype (GT) 3 {adjusted odds ratio [aOR] = 7.66 [95% of confidence interval (95% CI): 3.96 to 14.81] P < 0.001}, HCV-viremia <500,000 IU/mL [aOR = 2.20 (95% CI: 1.16 to 4.15] P = 0.015), no/mild liver fibrosis (F < 2) [aOR = 1.92 (95% CI: 1.08 to 3.42) P = 0.026], IL28B rs12980275 AA genotype [aOR = 2.70 (95% CI: 1.54 to 4.71) P < 0.001], and PPARγ2 rs1801282 CG/GG genotype [aOR = 2.93 (95% CI: 1.27 to 6.72) P = 0.011]. When PPARγ2 rs1801282 genotype was included in a decision tree analysis, HCV-GT3 patients with CG/GG genotype had increased SVR from 80.3% to 100%. In GT1/4 patients, rs12980275 AA carriers had increased SVR from 58.7% to 78.6%, and rs12980275 AG/GG carriers had increased SVR from 28.7% to 35.7%. The overall percentage of patients correctly classified was 71.6% and the area under the receiver operating characteristic curves was 0.766 ± 0.028. Conclusions: The presence of PPARγ2 rs1801282 G allele (Ala variant) was associated with increased odds for achieving SVR in HIV/HCV-coinfected patients on HCV treatment.
Plasma IL-6 and IL-9 predict the failure of interferon-α plus ribavirin therapy in HIV/HCV-coinfected patients.
(Oxford University Press, 2012-05) Guzman-Fulgencio, Maria; Jiménez, Jose Luis; Berenguer, Juan; Fernandez-Rodriguez, Amanda; López, Juan Carlos; Cosín, Jaime; Miralles, Pilar; Micheloud, Dariela; Muñoz-Fernández, Ma Ángeles; Resino, Salvador
Background: The cytokine profile plays an important role in treatment outcome of hepatitis C virus (HCV) infection, and probably modulates the immune response against HCV. The aim of this study was to evaluate which cytokines affect the response to interferon-α (IFN-α) and ribavirin therapy and how these cytokines change 72 weeks after starting anti-HCV therapy in HIV/HCV-coinfected patients. Methods: We carried out a retrospective follow-up study of 65 patients on anti-HCV therapy. A sustained virological response (SVR) was defined as an undetectable HCV viral load up to 24 weeks after the end of treatment. Cytokines were measured using a multiplex immunoassay kit. Results: On starting anti-HCV therapy, non-responder (NR) patients had higher levels of interleukin (IL)-6, IL-9, IL-10 and tumour necrosis factor (TNF)-α (P < 0.05), while IL-17A levels were increased in SVR patients (P = 0.058). However, only patients with high levels of IL-6 and IL-9 had decreased odds to achieve SVR (P < 0.05). Plasma levels of IL-6 and IL-9 had a high predictive value for SVR failure [area under the ROC curve (AUC) 0.839 (95% CI 0.733-0.945) and AUC 0.769 (95% CI 0.653-0.884)]. In addition, during anti-HCV therapy, IL-1β showed an increase in NR patients (P = 0.015) and IL-10 decreased in SVR patients (P = 0.049). After clearing HCV infection, low levels of TNF-α, IL-6, IL-9, IL-10, IL-13 and IL-22 were found in SVR patients (P < 0.05), as well as IL-1β, but only near statistical significance (P = 0.073). Conclusions: High plasma levels of IL-6 and IL-9 had a high predictive value for SVR failure. Furthermore, clearing of HCV infection was associated with low inflammatory and T helper (Th)2/Th9/Th22 cytokine levels.
Distribution of DNA gyrase cleavage sites across the Streptococcus pneumoniae genome: relation to transcription and methylation at GATC sites.
(Oxford University Press, 2025-11-13) Ferrandiz-Avellano, Maria-Jose; Hernández, Pablo; de la Campa, Adela G
In bacteria, DNA gyrase plays a key role in cellular processes by affecting supercoiling. However, how gyrase localizes on the chromosome to affect supercoiling is unclear. Here, we interrogate the genome-wide distribution of gyrase in Streptococcus pneumoniae by mapping its cleavage sites in the presence of fluoroquinolones. Fluoroquinolones induce the formation of DNA-fluoroquinolone-topoisomerase complexes and generate detrimental double-stranded DNA breaks. Using a chromatin immunoprecipitation-sequencing-based method, we localize gyrase cleavage sites with single-nucleotide resolution. A total of 1517 sites were detected; the majority (92.7%) were located within protein-coding genes and were associated with high-level transcription. This trend was maintained at the level of transcriptional topological domains, where the most transcribed genes exhibited the highest frequency of cleavage sites, while the least transcribed genes exhibited the lowest frequency. We identified a four-base motif, GxxC, in 79% of the sites. The most frequent sequence was GATC (21.2% of all cleavage sites). GATC is the target of three pneumococcal restriction systems: DpnI, DpnII, and DpnIII. We demonstrated that methylation at GATC by the DpnII (GAmeTC) or DpnIII (GATCme) systems decreased gyrase cleavage and supercoiling activity. We hypothesized that strains with methylated GATC would be more resistant to subinhibitory concentrations of fluoroquinolones. We indeed demonstrated that gyrase mutants appeared more frequently in strains with GATC methylation (DpnII and DpnIII) than in strains without methylation (DpnI).This is significant for the evolution of fluoroquinolone resistance in S. pneumoniae, as the three Dpn phenotypes are present in the population of clinical isolates.
Live attenuated vaccines, a favorable strategy to provide long-term immunity against protozoan diseases.
(Elsevier, 2022-04) Solana, Jose Carlos; Moreno, Javier; Iborra, Salvador; Soto, Manuel; Requena, Jose M
The control of diseases caused by protozoan parasites is one of the United Nations' Sustainable Development Goals. In recent years much research effort has gone into developing a new generation of live attenuated vaccines (LAVs) against malaria, Chagas disease and leishmaniasis. However, there is a bottleneck related to their biosafety, production, and distribution that slows downs further development. The success of irradiated or genetically attenuated sporozoites against malaria, added to the first LAV against leishmaniasis to be evaluated in clinical trials, is indicative that the drawbacks of LAVs are gradually being overcome. However, whether persistence of LAVs is a prerequisite for sustained long-term immunity remains to be clarified, and the procedures necessary for clinical evaluation of vaccine candidates need to be standardized.
Proteomics of plasma-derived extracellular vesicles from human patients identifies biomarkers for monitoring visceral leishmaniasis therapy.
(Frontiers Media, 2025) Torres Garcia, Ana Maria; Montero-Calle, Ana Maria; Lozano-Rendal, Marina; Sanchez Herrero, Carmen; Bernardo, Lorena; Solana, Jose Carlos; San Martin, Juan Victor; Barderas Manchado, Rodrigo; Moreno, Javier; Carrillo, Eugenia
Introduction: The most severe form of leishmaniasis, visceral leishmaniasis (VL), lacks standardized validated early predictors of treatment success or relapse. To distinguish between active infection and successful treatment, we searched for protein biomarkers in plasma-derived extracellular vesicles (EVs). Methods: The proteomic profiles of EVs from immunocompetent patients with active VL (n=12) or 1, 3, or 6 months after completing a standard treatment regimen (n=12 each) were analyzed by LC-MS/MS. Six candidate biomarkers were further tested by ELISA in whole plasma. Results: 132 human proteins were differentially expressed in active VL- versus successfully treated patients. Pathway analysis identified pathogenic mechanisms associated with VL and pathways related to effective cure. SAA is directly measurable in whole plasma and exhibits differential expression levels, emerging as a promising, easily measurable, non-specific prognostic biomarker for patient management. Remarkably, we also identified Leishmania spp. proteins in EV samples, indicating a new source of parasite biomarkers in human samples. Conclusion: Plasma EVs contain protein biomarkers that can be used to monitor the response to treatment, some of which are detectable in whole plasma after 1 month of treatment. Our study also provides a proteomic landscape of plasma EVs involved in VL, offering insight into the pathogenesis of this complex disease.
Diagnostic potential of GLP recombinant antigens in whole blood assays for Leishmania infantum infection.
(Springer, 2025-11-21) Ibarra-Meneses, Ana Victoria; Fernandez, Laura; San Martín, Juan Víctor; García-Martínez, Jesús; Moreno, Javier; Botana, Laura; Sanchez Herrero, Carmen; Solana, Jose Carlos; Bernardo, Lorena; Reed, Steven G; Coler, Rhea N; Moreno, Javier; Carrillo, Eugenia
Background: The whole blood stimulation assay (WBA) is a valuable tool for detecting asymptomatic Leishmania infection and monitoring the treatment of visceral leishmaniasis (VL). This study sought to identify specific recombinant proteins to replace the nonspecific soluble Leishmania antigen in this assay, which could be useful for developing a standardized diagnostic test that complies with good manufacturing practice. Methods: Employing a cell lymphoproliferative assay, we here assessed the behaviour of 11 recombinant antigens in 61 subjects who had either been successfully treated for or had spontaneously recovered from Leishmania infantum infection. We then selected those antigens showing significant differences in immune cell stimulation indices and cytokine secretion between a responder and non-responder group, respectively, showing a cellular response to L. infantum or not. The three best candidate antigens, ΔCpB, NSC and ENSC, were then used in a WBA conducted on peripheral blood from 53 subjects stratified according to leishmaniasis status [cured VL, cured cutaneous leishmaniasis (CL), asymptomatic leishmaniasis (AS) and healthy controls]. Results: ENSC was found to be the most effective antigen to detect cured VL by measuring specific IP-10 production (90% recognition) and TNF induced by ΔCpB to detect cured CL (71.4% recognition). Although the cytokines IL-2 and IP-10 elicited by NSC and ENSC were able to detect AS, this capacity was only moderate (60%). Conclusions: We propose that, once validated in larger studies, these GLP Leishmania antigens might help improve the accuracy of treatment monitoring and diagnosing cure.
Identification of Off-Patent Drugs That Show Synergism with Amphotericin B or That Present Antifungal Action against Cryptococcus neoformans and spp.
(American Society for Microbiology (ASM), 2020-03-24) Rossi, Suelen Andreia; de Oliveira, Haroldo Cesar; Agreda-Mellon, Daniel; Lucio, Jose; Mendes-Giannini, Maria José Soares; Garcia-Cambero, Jesus Pablo; Zaragoza, Oscar
Amphotericin B (AmB) is the antifungal with the strongest fungicidal activity, but its use has several limitations, mainly associated with its toxicity. Although some lipidic and liposomal formulations that present reduced toxicity are available, their price limits their application in developing countries. Flucytosine (5FC) has shown synergistic effect with AmB for treatment of some fungal infections, such as cryptococcosis, but again, its price is a limitation for its use in many regions. In the present work, we aimed to identify new drugs that have a minor effect on , reducing its growth in the presence of subinhibitory concentrations of AmB. In the initial screening, we found fourteen drugs that had this pattern. Later, checkerboard assays of selected compounds, such as erythromycin, riluzole, nortriptyline, chenodiol, nisoldipine, promazine, chlorcyclizine, cloperastine, and glimepiride, were performed and all of them confirmed for their synergistic effect (fractional inhibitory concentration index [FICI] < 0.5). Additionally, toxicity of these drugs in combination with AmB was tested in mammalian cells and in zebrafish embryos. Harmless compounds, such as the antibiotic erythromycin, were found to have synergic activity with AmB, not only against but also against some spp., in particular against In parallel, we identified drugs that had antifungal activity against and found 43 drugs that completely inhibited the growth of this fungus, such as ciclopirox and auranofin. Our results expand our knowledge about antifungal compounds and open new perspectives in the treatment of invasive mycosis based on repurposing off-patent drugs.

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