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Interferon (IFN)-α induced CD4+ T lymphopenia is a toxic effect of the treatment of chronic hepatitis C virus (HCV) in human immunodeficiency virus (HIV)-co-infected patients. To increase the knowledge about this secondary effect, we performed an analysis of the evolution of the T cell receptor excision circles (TRECs), CD4+ and CD8+ T cells and of their CD45RO+ and CD45RA+ subpopulations during the treatment of chronic hepatitis HCV with peginterferon alpha (pegIFN-α) + ribavirin. Twenty HCV/HIV-co-infected patients, with undetectable HIV load after highly active antiretroviral therapy (HAART), were treated with pegIFN-α + ribavirin. TRECs were determined using real-time polymerase chain reaction. CD4+ and CD8+ T cells and their CD45RO+ and CD45RA+ subpopulations were analysed by two-colour flow cytometry. Median baseline CD4+ and CD8+ T cells were 592 mm3 and 874 mm3, respectively. Median baseline CD45RO+ subpopulation was 48% for CD4+ T and 57% for CD8+ T lymphocytes. A progressive decrease in both T cell populations, as well as of their CD45RO+ and CD45RA+ subpopulations, was detected, with a difference between the baseline and nadir levels approaching 50%. The evolution of T cell populations and TRECs was independent of the response to the treatment. T lymphocytes and their subpopulations returned to baseline levels at 24 weeks after the end of treatment, with the exception of the T CD4+ CD45RA+ subpopulation. The ratio of CD4+ CD45RO+/CD4+ CD45RA+ increased from 0·89 (baseline) to 1·44 (24 weeks after the end of the therapy). TRECs/ml did not return to the basal values. In conclusion, a significant reduction of CD4+ and CD8+ T cells, and of their CD45RA+ and CD45RO+ subpopulations, in HIV/HCV co-infected patients treated with pegIFN-α was observed. Both subpopulations increased after the suppression of treatment, but the CD4+ CD45RA subpopulation did not reach the basal levels as a consequence, at least in part, of a decrease in thymic production. [ABSTRACT FROM AUTHOR]

Objectives: To describe and characterize the emergence of resistance to ceftolozane/tazobactam, ceftazidime/avibactam and imipenem/relebactam in a patient receiving ceftazidime/avibactam treatment for an MDR Pseudomonas aeruginosa CNS infection., Methods: One baseline (PA1) and two post-exposure (PA2 and PA3) isolates obtained before and during treatment of a nosocomial P. aeruginosa meningoventriculitis were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. The impact on β-lactam resistance of mutations in blaPDC and mexR was determined through cloning experiments and complementation assays., Results: Isolate PA1 showed baseline resistance mutations in DacB (I354A) and OprD (N142fs) conferring resistance to conventional antipseudomonals but susceptibility to ceftazidime/avibactam, ceftolozane/tazobactam and imipenem/relebactam. Post-exposure isolates showed two divergent ceftazidime/avibactam-resistant phenotypes associated with distinctive mutations affecting the intrinsic P PDC β-lactamase (S254Ins) (PA2: ceftolozane/tazobactam and ceftazidime/avibactam-resistant) or MexAB-OprM negative regulator MexR in combination with modification of PBP3 (PA3: ceftazidime/avibactam and imipenem/relebactam-relebactam-resistant). Cloning experiments demonstrated the role of PDC modification in resistance to ceftolozane/tazobactam and ceftazidime/avibactam. Complementation with a functional copy of the mexR gene in isolate PA3 restored imipenem/relebactam susceptibility., Conclusions: We demonstrated how P. aeruginosa may simultaneously develop resistance and compromise the activity of new β-lactam/β-lactamase inhibitor combinations when exposed to ceftazidime/avibactam through selection of mutations leading to PDC modification and up-regulation of MexAB-OprM-mediated efflux., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: To evaluate the serum expression of microRNAs (miRNAs) with ability to modulate the human immunodeficiency (HIV) replication or inflammatory status in people living with HIV (PLWH)., Methods: Forty healthy controls and two groups of PLWH were evaluated: (a) Group 1 ( n  = 30), patients with detectable viral load at inclusion, analyzed before receiving antiretroviral therapy (ART) and 12 months after initiating it; (b) Group 2 ( n  = 55), PLWH with prolonged undetectable viral load. Intestinal barrier disruption (I-FABP) and bacterial translocation (16S rDNA) markers, inflammatory markers such as interleukin (IL)-6 and sCD163, immune activation and expression of specific miRNAs were evaluated., Results: Serum concentrations of I-FABP, 16S rDNA, IL-6, sCD163 and activated T lymphocytes were increased in PLWH. Serum miR-34a was overexpressed at inclusion and remained elevated after ART. The expression of the remaining miRNAs that modulate HIV infectivity (miR-7, mir-29a, miR-150, and miR-223) was similar in PLWH and controls. Related to miRNAs implicated in inflammation (miR-21, miR-155, and miR-210), significant overexpression were observed in miR-21 and miR-210 levels in untreated PLWH, but levels were restored in those patients treated for a long period., Conclusion: A sustained overexpression of miR-34a was detected even after prolonged HIV controlled replication. miR-21 and miR-210 can be considered new markers of inflammation with high sensitivity to its modifications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cuesta-Sancho, Márquez-Ruiz, Illanes-Álvarez, Campaña-Gómez, Martín-Aspas, Trujillo-Soto, Romero, Galán, Rodríguez-Iglesias, Márquez-Coello and Girón-González.)

The aim of our study is to determine the discriminatory power of the Fourier transform infrared spectroscopy (FTIR), using multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) as molecular typing references. The study included seventeen isolates (OXA-23- and OXA-58-producing Acinetobacter baumannii) previously recovered from clinical specimens during the period May 2010-April 2011. Molecular typing was performed by PFGE and MLST. The specimens were analyzed in quadruplicate using the IR Biotyper (Bruker GmbH, Bremen, Germany). For each isolate, the average of the spectra was used for the analysis of the data. Comparing FTIR data with MLST, the results obtained by IR Biotyper are very consistent with those from MLST, since the software was able to differentiate the three ST assigned to the strains. Comparing FTIR data with PFGE, most results could be confirmed, as IR Biotyper clearly differentiated ST-80 SLV OXA-58-producing A. baumannii (pulsotype 3) from the rest of strains of OXA-58-producing A. baumannii (pulsotypes 1 and 2). All the OXA-23-producing A. baumannii isolates (pulsotype 4) grouped together by FTIR. FTIR proved to be an effective tool to investigate local epidemiology, and can achieve the same typeability and discriminatory power as genome-based methods., (© 2022. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Background: The aim of this study was to ascertain whether the extemporaneous technetium-99m radiopharmaceuticals - prepared by closed procedures - maintain or not sterility throughout their lifespan regardless the quality of the environmental air where they are prepared, stored and dispensed, provided that the basic aseptic rules for closed procedures are followed., Methods: Three different types of assays were performed in this study: 1) sterility tests of each and every one of the vials with the remains of technetium-99m radiopharmaceuticals, which have been prepared in our hospital radiopharmacy during three years without any special environmental air conditions; 2) integrity tests of punctured rubber plug closures using both microbial challenge testing and dye ingress testing; and 3) simulation of the dispensing process using a liquid growth medium., Results: Sterility tests of more than 6,000 vials with the remains of technetium-99m radiopharmaceuticals - prepared without any special ambient air conditions - were performed, all of them with a negative result (no growth of microorganisms occurs). The integrity of punctured rubber plugs closure was sufficiently proven under extreme conditions by two different methods. The maintenance of sterility during the dispensing process of technetium-99m radiopharmaceuticals was also proven by simulation of the dispensing process using a liquid growth medium., Conclusions: This study strongly supports that it is not necessary any special ambient air conditions to prepare, store and dispense extemporaneous technetium-99m radiopharmaceuticals in order to preserve their sterility when the basic aseptic rules for closed procedures are followed.

Background: The development of resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of Pseudomonas aeruginosa infections is concerning., Objectives: Characterization of the mechanisms leading to the development of OXA-10-mediated resistance to ceftolozane/tazobactam and ceftazidime/avibactam during treatment of XDR P. aeruginosa infections., Methods: Four paired ceftolozane/tazobactam- and ceftazidime/avibactam-susceptible/resistant isolates were evaluated. MICs were determined by broth microdilution. STs, resistance mechanisms and genetic context of β-lactamases were determined by genotypic methods, including WGS. The OXA-10 variants were cloned in PAO1 to assess their impact on resistance. Models for the OXA-10 derivatives were constructed to evaluate the structural impact of the amino acid changes., Results: The same XDR ST253 P. aeruginosa clone was detected in all four cases evaluated. All initial isolates showed OprD deficiency, produced an OXA-10 enzyme and were susceptible to ceftazidime, ceftolozane/tazobactam, ceftazidime/avibactam and colistin. During treatment, the isolates developed resistance to all cephalosporins. Comparative genomic analysis revealed that the evolved resistant isolates had acquired mutations in the OXA-10 enzyme: OXA-14 (Gly157Asp), OXA-794 (Trp154Cys), OXA-795 (ΔPhe153-Trp154) and OXA-824 (Asn143Lys). PAO1 transformants producing the evolved OXA-10 derivatives showed enhanced ceftolozane/tazobactam and ceftazidime/avibactam resistance but decreased meropenem MICs in a PAO1 background. Imipenem/relebactam retained activity against all strains. Homology models revealed important changes in regions adjacent to the active site of the OXA-10 enzyme. The blaOXA-10 gene was plasmid borne and acquired due to transposition of Tn6746 in the pHUPM plasmid scaffold., Conclusions: Modification of OXA-10 is a mechanism involved in the in vivo acquisition of resistance to cephalosporin/β-lactamase inhibitor combinations in P. aeruginosa., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Pseudomonas aeruginosa may develop resistance to novel cephalosporin/β-lactamase inhibitor combinations during therapy through the acquisition of structural mutations in AmpC., Objectives: To describe the molecular and biochemical mechanisms involved in the development of resistance to ceftolozane/tazobactam in vivo through the selection and overproduction of a novel AmpC variant, designated PDC-315., Methods: Paired susceptible/resistant isolates obtained before and during ceftolozane/tazobactam treatment were evaluated. MICs were determined by broth microdilution. Mutational changes were investigated through WGS. Characterization of the novel PDC-315 variant was performed through genotypic and biochemical studies. The effects at the molecular level of the Asp245Asn change were analysed by molecular dynamics simulations using Amber., Results: WGS identified mutations leading to modification (Asp245Asn) and overproduction of AmpC. Susceptibility testing revealed that PAOΔC producing PDC-315 displayed increased MICs of ceftolozane/tazobactam, decreased MICs of piperacillin/tazobactam and imipenem and similar susceptibility to ceftazidime/avibactam compared with WT PDCs. The catalytic efficiency of PDC-315 for ceftolozane was 10-fold higher in relation to the WT PDCs, but 3.5- and 5-fold lower for piperacillin and imipenem. IC50 values indicated strong inhibition of PDC-315 by avibactam, but resistance to cloxacillin inhibition. Analysis at the atomic level explained that the particular behaviour of PDC-315 is linked to conformational changes in the H10 helix that favour the approximation of key catalytic residues to the active site., Conclusions: We deciphered the precise mechanisms that led to the in vivo emergence of resistance to ceftolozane/tazobactam in P. aeruginosa through the selection of the novel PDC-315 enzyme. The characterization of this new variant expands our knowledge about AmpC-mediated resistance to cephalosporin/β-lactamase inhibitors in P. aeruginosa., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Human immunodeficiency virus (HIV) antibodies have been proposed as a measure of the size of the HIV reservoir. The aim of our study is to quantify the anti-HIV antibodies level in a cohort of people living with HIV (PLWH), stratified based on the presence of continuous undetectable HIV viral load and the co-existence of hepatitis C virus infection. A sample of 229 HIV-monoinfected (n = 114) or HIV/HCV-coinfected [either with resolved HCV infection (n = 75) or active HCV coinfection (n = 40)] patients, followed up a median of 34 (IQR 20-44) months, was studied. Anti-HIV index was obtained as the 1:800 dilution of HIV antibodies. CD4+ T cell count, time with undetectable HIV viral load, annual increase of CD4+ T cell count, anti-HCV therapy, and diagnosis of cirrhosis were analyzed. Patients with a continued suppressed HIV viral load had significant lower anti-HIV index compared with those with virologic failure during the follow-up. Significant higher CD4+ T cell increase was observed in those with a lower anti-HIV index. HIV-monoinfected patients showed an anti-HIV index significantly lower than patients with HCV coinfection. Resolved HCV infection after interferon-based therapy, but not with direct acting antivirals, was associated with a lower anti-HIV index. HIV/HCV-coinfected patients showed higher HIV antibodies level when compared with HIV-monoinfected individuals. A decrease in anti-HIV index in HIV/HCV-coinfected patients was detected when a sustained virological HCV response was obtained after interferon-based therapy, in possible relation with the direct or indirect effect of interferon on PLWH CD4 T cells.

Introduction: NDM-1 carbapenemase is spreading rapidly all over the world, but this metallo-beta-lactamase has just been detected for the first time in an Acinetobacter baumannii (Ab) isolate of the ST85 clone in Spain. The aim of this study was to characterize a NDM-1-producing carbapenem-resistant A. baumannii (CR-Ab) isolate submitted to the Andalusian PIRASOA [infection prevention program] referral laboratory., Methods: Carbapenemases were detected by PCR and Sanger DNA sequencing. Whole genome sequencing was performed by NGS (Miseq, Illumina). Resistance genes were identified with RESfinder, while MLSTfinder was used for sequence typing (ST). The genetic location of bla NDM-1 was determined by nuclease S-1/PFGE/hybridization with specific probe., Results: The isolate was susceptible to amikacin and tigecycline and belonged to the ST85 clone. bla OXA-94 and bla NDM-1 were identified by PCR and Sanger DNA sequencing, respectively. The resistance genes aadB, bla ADC-25 , bla NDM-1 , bla OXA-94 , msr(E), mph(E) and floR,sul2 were identified by NGS. The chromosome of the isolate contained a defective Tn125 transposon with bla NDM-1 flanked by the insertion sequences ISAbA125 and ISAba14. The bla NDM-1 gene was only detected in the chromosomal DNA., Conclusion: This is the first time that bla NDM-1 has been detected and characterized in a bla OXA-94 -producing CR-Ab isolate belonging to the ST85 clone in Spain., (Copyright © 2019 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Selection of extended-spectrum mutations in narrow-spectrum oxacillinases (e.g., OXA-2 and OXA-10) is an emerging mechanism for development of in vivo resistance to ceftolozane-tazobactam and ceftazidime-avibactam in Pseudomonas aeruginosa Detection of these challenging enzymes therefore seems essential to prevent clinical failure, but the complex phenotypic plasticity exhibited by this species may often lead to their underestimation. The underlying resistance mechanisms of two sequence type 175 (ST175) P. aeruginosa isolates showing multidrug-resistant phenotypes and recovered at early and late stages of a long-term nosocomial infection were evaluated. Whole-genome sequencing (WGS) was used to investigate resistance genomics, whereas molecular and biochemical methods were used for characterization of a novel extended-spectrum OXA-2 variant selected during therapy. The metallo-β-lactamase bla VIM-20 and the narrow-spectrum oxacillinase bla OXA-2 were present in both isolates, although they differed by an inactivating mutation in the mexB subunit, present only in the early isolate, and in a mutation in the bla OXA-2 β-lactamase, present only in the final isolate. The new OXA-2 variant, designated OXA-681, conferred elevated MICs of the novel cephalosporin-β-lactamase inhibitor combinations in a PAO1 background. Compared to OXA-2, kinetic parameters of the OXA-681 enzyme revealed a substantial increase in the hydrolysis of cephalosporins, including ceftolozane. We describe the emergence of the novel variant OXA-681 during treatment of a nosocomial infection caused by a Pseudomonas aeruginosa ST175 high-risk clone. The ability of OXA-681 to confer cross-resistance to ceftolozane-tazobactam and ceftazidime-avibactam together with the complex antimicrobial resistance profiles exhibited by the clinical strains harboring this new enzyme argue for maintaining active surveillance on emerging broad-spectrum resistance in P. aeruginosa ., (Copyright © 2019 American Society for Microbiology.)

Salmonella enterica is a well-adapted zoonotic bacterium associated to cases of gastroenteritis and bacteremia with increased morbidity and mortality. In this study, three isolates of Salmonella Typhimurium obtained from human clinical samples, showing colistin resistance and low-level resistance to quinolones, have been genetically characterized. We detected the co-occurrence of mcr-1 and qnrS1 on a single IncHI2 plasmid in isolates of Salmonella Typhimurium obtained from Spanish children without a travel history. The multiresistant region contained numerous resistance genes. Isolates were clonally related, which suggests the presence of these clones in the community and the potential to cause outbreaks affecting the most susceptible population. It is necessary to monitor the presence of these plasmid-mediated resistance genes in human European strains of Salmonella spp. because of the risk of producing outbreaks of community-acquired infections.

We analyzed the species distribution and susceptibility patterns of 433 strains of Aspergillus spp. isolated from respiratory samples of 419 in-patients included in multicenter prospective study (FUNGAE-IFI) between July 2014 and October 2015. Identification was carried out by conventional methods at each participating center and by molecular sequencing of a portion of the β-tubulin gene at one of the centers. In vitro susceptibility was evaluated by broth microdilution methods and using the E-test (for cryptic species). Species identified included 249 A. fumigatus sensu stricto, 60 A. terreus sensu stricto, 47 A. flavus sensu stricto, 44 A. tubingensis, 18 A. niger sensu stricto , five A. nidulans sensu stricto, three A. tamarii, two A. calidoustus, two A. carneus, one A. acuelatus, one A. carbonarius, and one A. sydowii. Cryptic species were found in 12.5% of isolates (n = 54). The frequency of non-wild-type isolates for amphotericin B was 3.4% (n = 15) of the isolates tested and for azoles 3% (n = 10). None of the Aspergillus spp. were non-wild type to echinocandins. Of the 54 cryptic species only two strains were non-wild-type strains by microdilution method (3.7%) (two A. tubingensis, one to amphotericin B and another one to voriconazole) and by E-test method five strains of A. tubingensis showed high minimal inhibitory concentration (MIC) to amphotericin B (11.4%) and five to azoles (12.1%), one A. calidoustus strain showed high MICs for three azoles (50%), A. carneus to itraconazole (100%) and A. sydowii to amphotericin B and itraconazole (100%). These results provide relevant information on susceptibility patterns, frequency, and epidemiology of species involved in respiratory tract samples and of the incidence of recently described cryptic species., (© The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The Human Papillomavirus (HPV) is a double-stranded DNA virus, with more than 200 different genotypes having been identified. This infection is considered the most common sexually transmitted infection (STI), and it is the cause of a significant number of diseases, both benign lesions (anogenital condylomas) and pre-malignant lesions and different cancers. The diagnosis of the infection is performed by molecular techniques based on the detection of viral DNA, the mRNA of oncogenic proteins and cellular alteration caused by the infection. Although there is no consensus regarding the best treatment, this should be individualised, and there are different options with ablative treatments being more effective but with greater recurrences, and immunomodulatory treatments being less effective in the short term but with fewer recurrences. Among the preventive strategies, vaccination against HPV is the best strategy against anogenital neoplasms and warts, its maximum effectiveness being when it is administered prior to exposure to HPV., (Copyright © 2019 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Introduction: The epidemiology of S. aureus depends on conditions in specific populations. Few studies of S. aureus colonization in the older population have been performed in Spain. The aim of this study was to determine the prevalence of methicillin-resistant S. aureus (MRSA) colonization and its molecular epidemiological characteristics in an institutionalized population in community residential care homes in Cadiz, Spain., Methods: A cross-sectional epidemiological study was conducted in three residential care homes for older people. Axilla and nostril samples were tested. Identification of S. aureus and antimicrobial susceptibility testing were by MALDI-TOF and MicroScan panels. MRSA strains were subjected to SCCmec typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The presence of Panton-Valentine leukocidin (PVL) genes was determined by PCR in all S. aureus strains., Results: A total of 293 residents were included. Fifty-one residents (17.4%) were colonized with methicillin-sensitive S. aureus (MSSA) and 11 (3.8%) with MRSA. Resistance to at least two aminoglycosides was observed in 25.4% of MSSA and 90.9% and of MRSA isolates, and resistance to levofloxacin in 80.3% of MSSA and 100% of MRSA isolates. SCCmecIV was detected in all isolates and all except one (ST-125) were ST-8. None of the S. aureus isolates were positive for PVL., Conclusions: A low rate of S. aureus carriage was detected and the prevalence of MRSA was very low. ST8-MRSA-IVc was the dominant clone, and only one strain belonged to ST125-MRSA-IVc. We found MRSA transmission within the residential care homes and a very high rate of quinolone resistance in MSSA and MRSA., (Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)