Reassessing HIV Detection Strategies: An Analysis of Opportunistic Screening vs. Indicator-Condition-Driven Diagnosis in Valencia, Spain.

Our study assessed the characteristics of people living with HIV (PLWH) detected via opportunistic screening in Valencia (Spain) to determine diagnoses potentially missed under a more restrictive, indicator-condition diagnostic strategy. We conducted a retrospective analysis of electronic health records of 97 PLWH diagnosed between April 2019 and August 2022. The main outcomes reported were patient CD4+ T cell count, known HIV risk factors at diagnosis, and missed opportunities for diagnosis, defined as the failure of a previously untested patient to undergo HIV testing despite attending previous visits to healthcare facilities prior to diagnosis. Successful linkage to care was achieved for 95.9% of diagnosed patients. Half of the PLWH were diagnosed late, while 47.8% did not meet the criteria for indicator-condition-driven HIV diagnosis at the time of their diagnosis. Additionally, 52.2% did not receive HIV testing despite an average of 5.1 ± 6.0 healthcare visits in the 12 months prior to diagnosis. Spaniards had more missed opportunities for diagnosis than foreigners (64% vs. 40%, p = 0.02). Depending solely on an indicator-condition-driven HIV diagnosis approach could result in 47.8% of cases being missed. Including "migrants" as a testing criterion could lower missed diagnoses to 25.3% but might create inequities in prevention access. In conclusion, our findings provide valuable insights to enhance HIV testing, early diagnosis, and linkage to care. While it is crucial to uphold the indicator-condition-driven HIV diagnosis as baseline practice, improving screening strategies will decrease late diagnoses and missed opportunities, thereby effectively contributing to end the epidemic.

In vitro activity of cefiderocol and other newly approved antimicrobials against multi-drug resistant Gram-negative pathogens recovered in intensive care units in Spain and Portugal / Actividad in vitro de cefiderocol y otros antimicrobianos recientemente aprobados frente a gramnegativos multirresistentes aislados en unidades de cuidados intensivos en España y Portugal

Introduction. The antimicrobial resistance is a significant public health threat, particularly for healthcare-associated infections caused by carbapenem-resistant Gram-negative pathogens which are increasingly reported worldwide. The aim of this study was to provide data on the in vitro antimicrobial activity of cefiderocol and that of commercially available comparator antibiotics against a defined collection of recent clinical multi-drug resistant (MDR) microorganisms, including carbapenem resistant Gram-negative bacteria collected from different regions in Spain and Portugal. Material and methods. A total of 477 clinical isolates of Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii and Stenotrophomonas maltophilia were prospectively (n=265) and retrospectively (n=212) included (2016-2019). Susceptibility testing was performed using standard broad microdilution and results were interpreted using CLSI-2021 and EUCAST-2021 criteria. Results. Overall, cefiderocol showed a good activity against Enterobacterales isolates, being 99.5% susceptible by CLSI and 94.5% by EUCAST criteria. It also demonstrated excellent activity against P. aeruginosa and S. maltophilia isolates, all being susceptible to this compound considering CLSI breakpoints. Regarding A. baumannii (n=64), only one isolate was resistant to cefiderocol. Conclusions. Our results are in agreement with other studies performed outside Spain and Portugal highlighting its excellent activity against MDR gram-negative bacteria. Cefiderocol is a therapeutic alternative to those available for the treatment of infections caused by these MDR bacteria. (AU)

Introducción. La resistencia a los antimicrobianos constituye una importante amenaza para la salud pública, especialmente en el caso de las infecciones relacionadas con la asistencia sanitaria causadas por patógenos gramnegativos resistentes a los carbapenémicos, las cuales están aumentando en todo el mundo. El objetivo de este estudio fue proporcionar datos sobre la actividad antimicrobiana in vitro de cefiderocol y la de antibióticos comparadores disponibles en el arsenal terapéutico frente a una colección definida de microorganismos multirresistentes (MDR) obtenidos de muestras clínicas, incluidas bacterias gramnegativas resistentes a carbapenemas procedentes de diferentes regiones de España y Portugal. Material y métodos. Se recogieron un total de 477 aislados clínicos de Enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii y Stenotrophomonas maltophilia de forma prospectiva (n=265) y retrospectiva (n=212) (2016-2019). El estudio de sensibilidad se realizó por microdilución standard y los resultados se analizaron empleando criterios del CLSI de 2021 y de EUCAST de 2021. Resultados. En general, cefiderocol demostró una buena actividad frente a aislados de Enterobacterales, siendo 99,5% sensible según criterios del CLSI y 94,5% según los de EUCAST. Cefiderocol demostró una excelente actividad frente a aislados de P. aeruginosa y S. maltophilia, siendo todos ellos sensibles a este compuesto considerando los puntos de corte del CLSI. En relación a A. baumannii (n=64), sólo un aislado fue resistente a cefiderocol. Conclusiones. Nuestros resultados concuerdan con los de otros estudios realizados fuera de España y Portugal en los que se destaca la excelente actividad de cefiderocol frente a bacterias gramnegativas MDR. Cefiderocol constituye una alternativa terapéutica a las disponibles en el tratamiento de las infecciones causadas por estos microorganismos. (AU)

Role of the first WHO mutation catalogue in the diagnosis of antibiotic resistance in Mycobacterium tuberculosis in the Valencia Region, Spain: a retrospective genomic analysis.

BACKGROUND: In June, 2021, WHO published the most complete catalogue to date of resistance-conferring mutations in Mycobacterium tuberculosis. Here, we aimed to assess the performance of genome-based antimicrobial resistance prediction using the catalogue and its potential for improving diagnostics in a real low-burden setting. METHODS: In this retrospective population-based genomic study M tuberculosis isolates were collected from 25 clinical laboratories in the low-burden setting of the Valencia Region, Spain. Culture-positive tuberculosis cases reported by regional public health authorities between Jan 1, 2014, and Dec 31, 2016, were included. The drug resistance profiles of these isolates were predicted by the genomic identification, via whole-genome sequencing (WGS), of the high-confidence resistance-causing variants included in the catalogue and compared with the phenotype. We determined the minimum inhibitory concentration (MIC) of the isolates with discordant resistance profiles using the resazurin microtitre assay. FINDINGS: WGS was performed on 785 M tuberculosis complex culture-positive isolates, and the WGS resistance prediction sensitivities were: 85·4% (95% CI 70·8-94·4) for isoniazid, 73·3% (44·9-92·2) for rifampicin, 50·0% (21·1-78·9) for ethambutol, and 57·1% (34·0-78·2) for pyrazinamide; all specificities were more than 99·6%. Sensitivity values were lower than previously reported, but the overall pan-susceptibility accuracy was 96·4%. Genotypic analysis revealed that four phenotypically susceptible isolates carried mutations (rpoB Leu430Pro and rpoB Ile491Phe for rifampicin and fabG1 Leu203Leu for isoniazid) known to give borderline resistance in standard phenotypic tests. Additionally, we identified three putative resistance-associated mutations (inhA Ser94Ala, katG Leu48Pro, and katG Gly273Arg for isoniazid) in samples with substantially higher MICs than those of susceptible isolates. Combining both genomic and phenotypic data, in accordance with the WHO diagnostic guidelines, we could detect two new multidrug-resistant cases. Additionally, we detected 11 (1·6%) of 706 isolates to be monoresistant to fluoroquinolone, which had been previously undetected. INTERPRETATION: We showed that the WHO catalogue enables the detection of resistant cases missed in phenotypic testing in a low-burden region, thus allowing for better patient-tailored treatment. We also identified mutations not included in the catalogue, relevant at the local level. Evidence from this study, together with future updates of the catalogue, will probably lead in the future to the partial replacement of culture testing with WGS-based drug susceptibility testing in our setting. FUNDING: European Research Council and the Spanish Ministerio de Ciencia.

Reporting antimicrobial susceptibilities and phenotypes of resistance to vancomycin in vancomycin-resistant Enterococcus spp. clinical isolates: A nationwide proficiency study / Informe de sensibilidad antimicrobiana y fenotipos de resistencia a vancomicina en aislados clínicos de Enterococcus spp. resistentes a vancomicina: estudio multicéntrico nacional

Introduction: The ability of Spanish microbiology laboratories to (a) determine antimicrobial susceptibility (AS), and (b) correctly detect the vancomycin resistance (VR) phenotype in vancomycin-resistant Enterococcus spp. (VRE) was evaluated. Methods: Three VRE isolates representing the VanA (E. faecium), VanB (E. faecium) and VanC (E. gallinarum) VR phenotypes were sent to 52 laboratories, which were asked for: (a) AS method used; (b) MICs of ampicillin, imipenem, vancomycin, teicoplanin, linezolid, daptomycin, ciprofloxacin, levofloxacin and quinupristin–dalfopristin, and high-level resistance to gentamicin and streptomycin; (c) VR phenotype. Results: (a) The most frequently used system was MicroScan; (b) according to the system, the highest percentage of discrepant MICs was found with gradient strips (21.3%). By antimicrobial, the highest rates of discrepant MICs ranged 16.7% (imipenem) to 0.7% (linezolid). No discrepant MICs were obtained with daptomycin or levofloxacin. Mayor errors (MEs) occurred with linezolid (1.1%/EUCAST) and ciprofloxacin (5.0%/CLSI), and very major errors (VMEs) with vancomycin (27.1%/EUCAST and 33.3%/CLSI) and teicoplanin (5.7%/EUCAST and 2.3%/CLSI). For linezolid, ciprofloxacin, and vancomycin, discrepant MICs were responsible for these errors, while for teicoplanin, errors were due to a misassignment of the clinical category. An unacceptable high percentage of VMEs was obtained using gradient strips (14.8%), especially with vancomycin, teicoplanin and daptomycin; (c) 86.4% of the centers identified VanA and VanB phenotypes correctly, and 95.0% the VanC phenotype. Conclusion: Most Spanish microbiology laboratories can reliably determine AS in VRE, but there is a significant percentage of inadequate interpretations (warning of false susceptibility) for teicoplanin in isolates with the VanB phenotype.(AU)

Introducción: Se evaluó la capacidad de los laboratorios de microbiología españoles para: (a) determinar la sensibilidad antimicrobiana (SA); y (b) detectar correctamente el fenotipo de resistencia a vancomicina (FRV) en Enterococcus spp. resistente a vancomicina (ERV). Métodos: Se enviaron 3 aislados de ERV (E. faecium/VanA, E. faecium/VanB y E. gallinarum/VanC) a 52 laboratorios, a los que se les solicitó: (a) método de SA; (b) CMI de ampicilina, imipenem, vancomicina, teicoplanina, linezolid, daptomicina, ciprofloxacino, levofloxacino y quinupristina-dalfopristina y resistencia de alto nivel a gentamicina y estreptomicina; y (c) fenotipo de resistencia a vancomicina. Resultados: (a) El sistema más utilizado fue MicroScan; y (b) el mayor porcentaje de CMI discrepantes se produjo con las tiras de gradiente (21,3%). Las tasas más elevadas de CMI discrepantes variaron entre el 16,7% (imipenem) y el 0,7% (linezolid). Se produjeron errores mayores con linezolid (1,1%/EUCAST) y ciprofloxacino (5,0%/CLSI) y errores máximos con vancomicina (27,1%/EUCAST y 33,3% CLSI) y teicoplanina (5,7%/EUCAST y 2,3%/CLSI). Para linezolid, ciprofloxacino y vancomicina las CMI discrepantes fueron las responsables de estos errores, mientras que para teicoplanina los errores se debieron a una asignación errónea de la categoría clínica. Se obtuvo un alto porcentaje de errores máximos utilizando tiras de gradiente (14,8%), especialmente con vancomicina, teicoplanina y daptomicina; y (c) el 86,4% de los centros identificaron correctamente los fenotipos VanA y VanB y el 95,0% el fenotipo VanC. Conclusión: La mayoría de los laboratorios de microbiología españoles determinan de forma fiable la SA en ERV, pero existe un porcentaje significativo de interpretaciones inadecuadas (falsa sensibilidad) para teicoplanina en aislados con fenotipo VanB.(AU)

Genomic analysis of the initial dissemination of carbapenem-resistant Klebsiella pneumoniae clones in a tertiary hospital.

Carbapenem-resistant Klebsiella pneumoniae is a major cause of hospital-acquired infections and the fastest-growing pathogen in Europe. Carbapenem resistance was detected at the Consorcio Hospital General Universitario de Valencia (CHGUV) in early 2015, and there has been a significant increase in carbapenem-resistant isolates since then. In this study, we collected carbapenem-resistant isolates from this hospital during the period of increase (from 2015 to 2019) and studied how K. pneumoniae carbapenem-resistant isolates emerged and spread in the hospital. A total of 225 isolates were subjected to whole-genome sequencing with Illumina NextSeq. We characterized the isolates by identifying lineages and antimicrobial resistance genes and plasmids, especially those related to reduced carbapenem susceptibility. Our findings show that the initial carbapenem resistance emergence and dissemination at the CHGUV occurred during a short period of 1 year. Furthermore, it was complex, involving six different lineages of types ST307, ST11, ST101 and ST437, different resistance-determinant factors, including OXA-48, NDM-1, NDM-23 and DHA-1, and different plasmids.

Tracking the Emergence and Dissemination of a blaNDM-23 Gene in a Multidrug Resistance Plasmid of Klebsiella pneumoniae.

Since the discovery of blaNDM-1, NDM ß-lactamases have become one of the most widespread carbapenemases worldwide. To date, 43 different NDM variants have been reported but some, such as blaNDM-23, have not been characterized in detail yet. Here, we describe the emergence of a novel blaNDM-23 allele from a blaNDM-1 ancestor and the multidrug resistance plasmid that has disseminated it through a Klebsiella pneumoniae ST437 clone in several Spanish hospitals. Between 2016 and 2019, 1,972 isolates were collected in an epidemiological survey for extended-spectrum-ß-lactamase (ESBL)-producing Klebsiella pneumoniae in the Comunitat Valenciana (Spain). Three carbapenem-resistant strains failed to be detected by carbapenemase-producing Enterobacteriaceae (CPE) screening tests. These isolates carried a blaNDM-23 gene. To characterize this gene, its emergence, and its dissemination, we performed antimicrobial susceptibility tests, hybrid sequencing with Illumina and Nanopore technologies, and phylogenetic analyses. The MICs of the blaNDM-23 allele were identical to those of the blaNDM-1 allele. The blaNDM-23 allele was found in 14 isolates on a 97-kb nonmobilizable, multidrug-resistant plasmid carrying 19 resistance genes for 9 different antimicrobial families. In this plasmid, the blaNDM-23 gene is in the variable region of a complex class 1 integron with a singular genetic environment. The small genetic distance between blaNDM-23-producing isolates reflects a 5-year-long clonal dispersion involving several hospitals and interregional spread. We have characterized the genomic and epidemiological contexts in the emergence and community spread of a new blaNDM-23 allele in a multidrug resistance (MDR) plasmid of Klebsiella pneumoniae. IMPORTANCE At a time when antimicrobial resistance has become one of the biggest concerns worldwide, the emergence of novel alleles and extremely drug-resistant plasmids is a threat to public health worldwide, especially when they produce carbapenem resistance in one of the most problematic pathogens, such as Klebsiella pneumoniae. We used genomic epidemiology to describe the emergence of a novel NDM-23 allele and identify it in a MDR plasmid that has disseminated through a K. pneumoniae ST437 clone in several hospitals in Spain. Using bioinformatic and phylogenetic analyses, we have traced the evolutionary and epidemiological route of the new allele, the hosting plasmid, and the strain that carried both of them from Pakistan to Spain. A better understanding of the NDM-producing K. pneumoniae populations and plasmids has made evident the spread of this clone through the region, enhancing the importance of genomic surveillance in the control of antimicrobial resistance.

Reporting antimicrobial susceptibilities and phenotypes of resistance to vancomycin in vancomycin-resistant Enterococcus spp. clinical isolates: A nationwide proficiency study.

INTRODUCTION: The ability of Spanish microbiology laboratories to (a) determine antimicrobial susceptibility (AS), and (b) correctly detect the vancomycin resistance (VR) phenotype in vancomycin-resistant Enterococcus spp. (VRE) was evaluated. METHODS: Three VRE isolates representing the VanA (E. faecium), VanB (E. faecium) and VanC (E. gallinarum) VR phenotypes were sent to 52 laboratories, which were asked for: (a) AS method used; (b) MICs of ampicillin, imipenem, vancomycin, teicoplanin, linezolid, daptomycin, ciprofloxacin, levofloxacin and quinupristin-dalfopristin, and high-level resistance to gentamicin and streptomycin; (c) VR phenotype. RESULTS: (a) The most frequently used system was MicroScan; (b) according to the system, the highest percentage of discrepant MICs was found with gradient strips (21.3%). By antimicrobial, the highest rates of discrepant MICs ranged 16.7% (imipenem) to 0.7% (linezolid). No discrepant MICs were obtained with daptomycin or levofloxacin. Mayor errors (MEs) occurred with linezolid (1.1%/EUCAST) and ciprofloxacin (5.0%/CLSI), and very major errors (VMEs) with vancomycin (27.1%/EUCAST and 33.3%/CLSI) and teicoplanin (5.7%/EUCAST and 2.3%/CLSI). For linezolid, ciprofloxacin, and vancomycin, discrepant MICs were responsible for these errors, while for teicoplanin, errors were due to a misassignment of the clinical category. An unacceptable high percentage of VMEs was obtained using gradient strips (14.8%), especially with vancomycin, teicoplanin and daptomycin; (c) 86.4% of the centers identified VanA and VanB phenotypes correctly, and 95.0% the VanC phenotype. CONCLUSION: Most Spanish microbiology laboratories can reliably determine AS in VRE, but there is a significant percentage of inadequate interpretations (warning of false susceptibility) for teicoplanin in isolates with the VanB phenotype.

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in a Tertiary Hospital from the Comunidad Valenciana (Spain).

To reduce the high rates of morbidity and mortality caused by methicillin-resistant Staphylococcus aureus (MRSA) strains, it is essential to prevent their transmission. This can be achieved through molecular surveillance of the infecting strains, for which the detection of the entry of new strains, the analysis of antimicrobial resistance, and their containment are essential. In this study, we have analyzed 190 MRSA isolates obtained at the Consorcio Hospital General Universitario de Valencia (Spain) from 2013 to 2018 with three approaches: Multilocus Sequence Typing, spa, and SCCmec typing. Although the incidence of S. aureus infections detected in the hospital increased in the study period, the frequency of MRSA isolates decreased from 33% to 18%. One hundred seventy-two MRSA isolates were resistant to three or more classes of antimicrobials, especially to fluoroquinolones. No relevant temporal trend in the distribution of antibiotic susceptibility was observed. The combination of the three typing schemes allowed the identification of 74 different clones, of which the combination ST125-t067-IV was the most abundant in the study (27 cases). Members of three clonal complexes, CC5, CC8, and CC22, comprised 91% of the isolates, and included 32 STs and 32 spa types. The emergence of low incidence strains throughout the study period and a large number of isolates resistant to different classes of antibiotics shows the need for epidemiological surveillance of this pathogen. Our study demonstrates that epidemiological and molecular surveillance is a powerful tool to detect the emergence of clinically important MRSA clones.

Population-based sequencing of Mycobacterium tuberculosis reveals how current population dynamics are shaped by past epidemics.

Transmission is a driver of tuberculosis (TB) epidemics in high-burden regions, with assumed negligible impact in low-burden areas. However, we still lack a full characterization of transmission dynamics in settings with similar and different burdens. Genomic epidemiology can greatly help to quantify transmission, but the lack of whole genome sequencing population-based studies has hampered its application. Here, we generate a population-based dataset from Valencia region and compare it with available datasets from different TB-burden settings to reveal transmission dynamics heterogeneity and its public health implications. We sequenced the whole genome of 785 Mycobacterium tuberculosis strains and linked genomes to patient epidemiological data. We use a pairwise distance clustering approach and phylodynamic methods to characterize transmission events over the last 150 years, in different TB-burden regions. Our results underscore significant differences in transmission between low-burden TB settings, i.e., clustering in Valencia region is higher (47.4%) than in Oxfordshire (27%), and similar to a high-burden area as Malawi (49.8%). By modeling times of the transmission links, we observed that settings with high transmission rate are associated with decades of uninterrupted transmission, irrespective of burden. Together, our results reveal that burden and transmission are not necessarily linked due to the role of past epidemics in the ongoing TB incidence, and highlight the need for in-depth characterization of transmission dynamics and specifically tailored TB control strategies.

SARS-CoV-2 Delta-variant breakthrough infections in nursing home residents at midterm after Comirnaty® COVID-19 vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta variant breakthrough infections in nursing home residents following vaccination with Comirnaty® COVID-19 vaccine were characterized. In total, 201 participants (median age, 87 years; range, 64-100; 133 female) from two nursing homes in the Valencian community (Spain) were included. SARS-CoV-2-Spike (S) antibody responses were determined by a lateral flow immunocromatography (LFIC) assay and by quantitative electrochemiluminescent assay in LFIC-negative participants. SARS-CoV-2-S-IFNγ T cells were enumerated by flow cytometry in 10 participants. Nasopharyngeal SARS-CoV-2 RNA loads were quantified by real-time polymerase chain reaction assays. Vaccine breakthrough COVID-19 due to the Delta variant occurred in 39 residents (median age, 87 years; range, 69-96; 31 female) at a median of 6.5 months after vaccination (nine requiring hospitalization). Breakthrough infections occurred at a higher rate (p < 0.0001) in residents who had not been previously infected with SARS-CoV-2 (naïve) (33/108; 18%) than in those with prior diagnosis of SARS-CoV-2 infection (experienced) (6/93; 6.4%), and were more likely (p < 0.0001) to develop in residents who tested negative by LFIC (20/49) at 3 months after vaccination as compared to their LFIC-positive counterparts (19/142). Among LFIC-negative residents, a trend towards lower plasma anti-RBD antibody levels was noticed in those developing breakthrough infection (p = 0.16). SARS-CoV-2 RNA loads in nasopharyngeal specimens were lower in SARS-CoV-2-experienced residents (p < 0.001) and in those testing positive by LFIC (p = 0.13). The frequency of SARS-CoV-2-S-reactive T cells at 3 months was similar in LFIC-negative residents with (n = 7) or without (n = 3) breakthrough infection. Prior history of SARS-CoV-2 infection and detection of S-reactive antibodies by LFIC at 3 months is associated with a lower risk of Delta-variant breakthrough infection in nursing home residents at midterm after Comirnaty® COVID-19 vaccination.

Performance comparison of a flow cytometry immunoassay for intracellular cytokine staining and the QuantiFERON® SARS-CoV-2 test for detection and quantification of SARS-CoV-2-Spike-reactive-IFN-γ-producing T cells after COVID-19 vaccination.

PURPOSE: We compared the performance of an in-house-developed flow cytometry assay for intracellular cytokine staining (FC-ICS) and a commercially-available cytokine release assay (the QuantiFERON® SARS-CoV-2 Test [QF]) for detection and quantification of SARS-CoV-2-Spike (S)-reactive-IFN-γ-producing T cells after COVID-19 vaccination. PATIENTS AND METHODS: The sample included 141 individuals (all male; median age, 42 years; 20-72) who had been fully vaccinated with the Comirnaty® COVID-19 vaccine (at a median of 114 days; 34-145). Prior to vaccination, 91 were categorized as being SARS-CoV-2-naïve and 50 as SARS-CoV-2-experienced. A whole blood-based FC-ICS using 15-mer overlapping peptides encompassing the entire SARS-CoV-2 S protein was used for enumeration of virus-specific IFN-γ-producing CD4+ and CD8+ T cells. The QF test (Ag1 for CD4+ T cells and Ag2 for CD4+ and CD8+ T cells in combination) was carried out following the manufacturer's instructions. RESULTS: The FC-ICS and the QF assays returned significantly discordant qualitative results in both the entire cohort (P<0.001 with QF Ag1 and QF Ag2) and in SARS-CoV-2-naïve participants alone (P=0.005 and P=0.01, respectively). Discrepant results mostly involved FC-ICS positive/QF negative specimens. Overall, no correlation was found either between SARS-CoV-2 IFN-γ- CD4+ T-cell frequencies and IFN-γ levels measured in the QF Ag1 tube (P=0.78) or between the sum of SARS-CoV-2 IFN-γ CD4+ and CD8+ T-cell frequencies and IFN-γ levels quantified in the QF Ag2 tube. CONCLUSION: The data suggest a greater sensitivity for the FC-ICS assay than the QF test, and urge caution when comparing SARS-CoV-2 T-cell immune responses assessed using different analytical platforms.

Commercial Interferon-gamma release assay to assess the immune response to first and second doses of mRNA vaccine in previously COVID-19 infected versus uninfected individuals.

We analysed immunological response during vaccination by using quantitative anti-spike IgG antibodies (qAbs) and Interferon-gamma (IFNγ) production by SARS-CoV-2-specific CD4+ and CD8+ T cells (QuantiFERON® assay). Blood samples were collected at four time points: a day before the reception of first (T0) and second (T1) BNT162b2 doses, 14 (T2) and 28 days (T3) after second dose. Fifty individuals were included: 34 previously infected by SARS-CoV-2 (CoV2+) and 16 that were not (CoV2-). Among CoV2+, we only observed significant differences after the first dose in both qAbs and IFNγ+ T cells. CoV2- showed differences after each dose, and the response was lower than CoV2+. Older people presented a higher response in CoV2+, while in CoV2, young people responded best. Our results suggest that the second BNT162b2 vaccine dose is not a priority in people with previous COVID-19. QuantiFERON® is a good option to monitor T-cell immunity to SARS-CoV-2.

Contamination of tissue allografts from a multiorgan-multitissue donor colonized by Candida auris.

Standards on tissue banking determine the need of microbiological monitoring during critical steps (recovery, processing, storage, and transplantation). This information will be useful for both discarding contaminated tissues or risk analysis (in case of recipient infection). In this study, we show the case of a multiorgan-multitissue donor colonized by Candida auris. This microorganism is characterized by multidrug resistance, with higher transmissibility and severe outcome. Some of the microbiological cultures from arteries tested positive for this microorganism, but it was not cultured in samples from musculoskeletal tissues and corneas. No recipient case of infection transmission by Candida species was observed (organs and cornea). The implementation of active surveillance protocol for C. auris detection in critical care units (as source of tissue donors) has been suggested as a part of our hospitals' infection control policy.

Evaluation of a mass spectrometry and Vitek 2 combined protocol for rapid identification and susceptibility testing of Enterobacterales directly from positive blood cultures / Evaluación de la combinación de espectrometría de masas y el sistema Vitek 2 para la identificación rápida y el estudio de sensibilidad de Enterobacterales directamente desde frascos de hemocultivo positivos

OBJECTIVE: The aim was to evaluate a rapid method which would combine identification and susceptibility testing directly from positive blood cultures for Gram-negative bacilli of the Enterobacterales. MATERIAL AND METHODS: Gram-negative rods from blood cultures were directly identified by MALDI-TOF. Samples with Enterobacterales were selected for direct antimicrobial susceptibility testing by Vitek 2. The results were compared to those obtained with our laboratory's standard method. RESULTS: MALDI-TOF directly from blood cultures identified correctly 83% of the samples. Enterobacterales (n = 68) were identified at gender and species level in 85% of blood cultures with a score >1.7. In general, MICs were obtained after 7 h. MICs of amoxicillin-clavulanate, amikacin and ciprofloxacin showed in almost 50% of the cases after 5 h. CONCLUSIONS: A simple procedure with low cost and reduced working time makes it possible to integrate both identification and susceptibility testing directly from blood cultures. Thus, this protocol could offer advantages when it comes to selection and cost of treatment and patients' clinical outcomes

OBJETIVO: Evaluar un método rápido de identificación y estudio de sensibilidad directamente desde hemocultivos positivos para bacilos gramnegativos del orden Enterobacterales. MATERIAL Y MÉTODOS: Los hemocultivos con bacilos gramnegativos fueron utilizados para identificación mediante MALDI-TOF, seleccionándose aquellos con Enterobacterales para estudio de sensibilidad con Vitek 2. Los resultados fueron comparados con el método estándar del laboratorio. RESULTADOS: MALDI-TOF identificó correctamente el 83% de las muestras obtenidas directamente de hemocultivos. En caso de Enterobacterales (n = 68), el 85% se identificaron a nivel de género y especie con un score > 1,7. El tiempo necesario para la obtención de CMIs fue de 7 horas, reduciéndose a 5 en amoxicilina-clavulánico, amicacina y ciprofloxacino, casi en el 50% de los casos. CONCLUSIONES: Un protocolo simplificado, con bajo coste y reducido tiempo de trabajo, combinando identificación y estudio de sensibilidad directamente desde hemocultivos, podría proporcionar beneficios en la elección y coste del tratamiento, y mejora clínica del paciente

Molecular epidemiology and drug-resistance mechanisms in carbapenem-resistant Klebsiella pneumoniae isolated in patients from a tertiary hospital in Valencia, Spain.

OBJECTIVES: The aim of this study has been to characterize carbapenem-resistant Klebsiella pneumoniae isolates and to determine the resistance mechanisms involved, the clonal relationship between strains and clinical and demographical data of the infected patients. METHODS: Clinical and demographical data from patients were collected and statistically analysed. Antimicrobial susceptibility testing was performed and resistance genes were detected both phenotypically and genotypically. Conjugation assays were performed to show horizontal transferability of resistance genes. Clonal relationship was also studied. Next-generation sequencing (NGS) was performed to obtain information regarding resistance genes, sequence types, virulence factors and plasmid types. RESULTS: Statistical significance was shown by the presence of an infection if there had been a previous hospital stay; urinary catheter carriage and chronic renal disease also indicated higher probabilities of being infected. More than 95% of the isolates were non-susceptible to third-generation cephalosporins, and more than 90% were non-susceptible to quinolones. Phenotypic and genotypic methods for resistance detection were concordant and later confirmed by NGS. This is the first detection of OXA-48, NDM-1 and CTX-M-15 co-production in the area. No plasmid-mediated colistin resistance was found. Tetracycline, sulfonamides and aminoglycoside resistance genes were found in almost all the isolates studied. No virulence factors were detected. Multilocus sequence typing showed more than 15 different sequence types, with ST101, ST307 and ST11 being the most prevalent. CONCLUSIONS: This study is the first to report such a large group of OXA-48 carbapenemases with clonal dissemination among carbapenem-resistant K. pneumoniae in Valencia. This is also the first detection of OXA-48, NDM-1 and CTX-M-15 co-production in the area.

Evaluation of a mass spectrometry and Vitek 2 combined protocol for rapid identification and susceptibility testing of Enterobacterales directly from positive blood cultures.

OBJECTIVE: The aim was to evaluate a rapid method which would combine identification and susceptibility testing directly from positive blood cultures for Gram-negative bacilli of the Enterobacterales. MATERIAL AND METHODS: Gram-negative rods from blood cultures were directly identified by MALDI-TOF. Samples with Enterobacterales were selected for direct antimicrobial susceptibility testing by Vitek 2. The results were compared to those obtained with our laboratory's standard method. RESULTS: MALDI-TOF directly from blood cultures identified correctly 83% of the samples. Enterobacterales (n=68) were identified at gender and species level in 85% of blood cultures with a score >1.7. In general, MICs were obtained after 7h. MICs of amoxicillin-clavulanate, amikacin and ciprofloxacin showed in almost 50% of the cases after 5h. CONCLUSIONS: A simple procedure with low cost and reduced working time makes it possible to integrate both identification and susceptibility testing directly from blood cultures. Thus, this protocol could offer advantages when it comes to selection and cost of treatment and patients' clinical outcomes.

High-resolution mapping of tuberculosis transmission: Whole genome sequencing and phylogenetic modelling of a cohort from Valencia Region, Spain.

BACKGROUND: Whole genome sequencing provides better delineation of transmission clusters in Mycobacterium tuberculosis than traditional methods. However, its ability to reveal individual transmission links within clusters is limited. Here, we used a 2-step approach based on Bayesian transmission reconstruction to (1) identify likely index and missing cases, (2) determine risk factors associated with transmitters, and (3) estimate when transmission happened. METHODS AND FINDINGS: We developed our transmission reconstruction method using genomic and epidemiological data from a population-based study from Valencia Region, Spain. Tuberculosis (TB) incidence during the study period was 8.4 cases per 100,000 people. While the study is ongoing, the sampling frame for this work includes notified TB cases between 1 January 2014 and 31 December 2016. We identified a total of 21 transmission clusters that fulfilled the criteria for analysis. These contained a total of 117 individuals diagnosed with active TB (109 with epidemiological data). Demographic characteristics of the study population were as follows: 80/109 (73%) individuals were Spanish-born, 76/109 (70%) individuals were men, and the mean age was 42.51 years (SD 18.46). We found that 66/109 (61%) TB patients were sputum positive at diagnosis, and 10/109 (9%) were HIV positive. We used the data to reveal individual transmission links, and to identify index cases, missing cases, likely transmitters, and associated transmission risk factors. Our Bayesian inference approach suggests that at least 60% of index cases are likely misidentified by local public health. Our data also suggest that factors associated with likely transmitters are different to those of simply being in a transmission cluster, highlighting the importance of differentiating between these 2 phenomena. Our data suggest that type 2 diabetes mellitus is a risk factor associated with being a transmitter (odds ratio 0.19 [95% CI 0.02-1.10], p < 0.003). Finally, we used the most likely timing for transmission events to study when TB transmission occurred; we identified that 5/14 (35.7%) cases likely transmitted TB well before symptom onset, and these were largely sputum negative at diagnosis. Limited within-cluster diversity does not allow us to extrapolate our findings to the whole TB population in Valencia Region. CONCLUSIONS: In this study, we found that index cases are often misidentified, with downstream consequences for epidemiological investigations because likely transmitters can be missed. Our findings regarding inferred transmission timing suggest that TB transmission can occur before patient symptom onset, suggesting also that TB transmits during sub-clinical disease. This result has direct implications for diagnosing TB and reducing transmission. Overall, we show that a transition to individual-based genomic epidemiology will likely close some of the knowledge gaps in TB transmission and may redirect efforts towards cost-effective contact investigations for improved TB control.

Emergence of NDM-producing Klebsiella pneumoniae and Escherichia coli in Spain: phylogeny, resistome, virulence and plasmids encoding blaNDM-like genes as determined by WGS.

OBJECTIVES: NDM carbapenemases have spread worldwide. However, little information exists about the impact of NDM-producing Enterobacteriaceae in Spain. By WGS, we sought to elucidate the population structure of NDM-like-producing Klebsiella pneumoniae and Escherichia coli in Spain and to determine the plasmids harbouring blaNDM-like genes. METHODS: High-resolution SNP typing, core-genome MLST and plasmid reconstruction (PlasmidID) were performed on 59 NDM-like-producing K. pneumoniae and 8 NDM-like-producing E. coli isolated over an 8 year period in Spain. RESULTS: Five major epidemic clones of NDM-producing K. pneumoniae caused five important nationwide outbreaks: ST437/NDM-7, ST437/NDM-1, ST147/NDM-1, ST11/NDM-1 and ST101/NDM-1; in contrast, the spread of NDM-producing E. coli was polyclonal. Three blaNDM types were identified: blaNDM-1, 61.2%; blaNDM-7, 32.8%; and blaNDM-5, 6%. Five K. pneumoniae isolates co-produced other carbapenemases (three blaOXA-48 and two blaVIM-1). The average number of acquired resistance genes was higher in K. pneumoniae than in E. coli. The plasmids encoding blaNDM-like genes belonged to IncFII, IncFIB, IncX3, IncR, IncN and IncC types, of which IncF, IncR and IncC were associated with MDR. The genetic surroundings of blaNDM-like genes showed a highly variable region upstream of ISAba125. CONCLUSIONS: In recent years NDM-producing K. pneumoniae and E. coli have emerged in Spain; the spread of a few high-risk K. pneumoniae clones such as ST437/NDM-7, ST437/NDM-1, ST147/NDM-1, ST11/NDM-1 and ST101/NDM-1 have caused several interregional outbreaks. In contrast, the spread of NDM-producing E. coli has been polyclonal. Plasmid types IncFII, IncFIB, IncX3, IncR, IncN and IncC carried blaNDM, and the same IncX3 plasmid was detected in K. pneumoniae and E. coli.

A survey on practices for active surveillance of carriage of multidrug-resistant bacteria in hospitals in the Autonomous Community of Valencia, Spain.

A questionnaire-based cross-sectional study was conducted to gather information on current microbiological practices for active surveillance of carriage of multidrug-resistant (MDR) bacteria in hospitals from 14 health departments of the Autonomous Community of Valencia (ACV), Spain, which together provided medical attention to 3,271,077 inhabitants in 2017, approximately 70% of the population of the ACV. The survey consisted of 35 questions on MDR bacteria screening policies, surveillance approach chosen (universal vs. targeted), and microbiological methods and processes in use for routine detection and reporting of colonization by MDR bacteria, including the anatomical sites scheduled to be sampled for each MDR bacterial species, and the methodology employed (culture-based, molecular-based, or both). Our study revealed striking differences across centers, likely attributable to the lack of consensus on optimal protocols for sampling, body sites for screening, and microbiological testing, thus underscoring the need for consensus guidelines on these issues.