Genomic Surveillance Uncovers a 10-Year Persistence of an OXA-24/40 Acinetobacter baumannii Clone in a Tertiary Hospital in Northern Spain.

Infections caused by carbapenem-resistant Acinetobacter baumannii are a global threat causing a high number of fatal infections. This microorganism can also easily acquire antibiotic resistance determinants, making the treatment of infections a big challenge, and has the ability to persist in the hospital environment under a wide range of conditions. The objective of this work was to study the molecular epidemiology and genetic characteristics of two blaOXA24/40Acinetobacter baumannii outbreaks (2009 and 2020-21) at a tertiary hospital in Northern Spain. Thirty-six isolates were investigated and genotypically screened by Whole Genome Sequencing to analyse the resistome and virulome. Isolates were resistant to carbapenems, aminoglycosides and fluoroquinolones. Multi-Locus Sequence Typing analysis identified that Outbreak 1 was mainly produced by isolates belonging to ST3Pas/ST106Oxf (IC3) containing blaOXA24/40, blaOXA71 and blaADC119. Outbreak 2 isolates were exclusively ST2Pas/ST801Oxf (IC2) blaOXA24/40, blaOXA66 and blaADC30, the same genotype seen in two isolates from 2009. Virulome analysis showed that IC2 isolates contained genes for capsular polysaccharide KL32 and lipooligosacharide OCL5. A 8.9 Kb plasmid encoding the blaOXA24/40 gene was common in all isolates. The persistance over time of a virulent IC2 clone highlights the need of active surveillance to control its spread.

Genotypic and Phenotypic Study of Antiviral Resistance Mutations in Refractory Cytomegalovirus Infection.

BACKGROUND: This study describes the genotypic and phenotypic characterization of novel human cytomegalovirus (HCMV) genetic variants of a cohort of 94 clinically resistant HCMV patients. METHODS AND RESULTS: Antiviral-resistant mutations were detected in the UL97, UL54, and UL56 target genes of 25 of 94 (26.6%) patients. The genotype-phenotype correlation study resolved the status of 5 uncharacterized UL54 deoxyribonucleic acid polymerase (G441S, A543V, F460S, R512C, A928T) and 2 UL56 terminase (F345L, P800L) mutations found in clinical isolates. A928T conferred high, triple resistance to ganciclovir, foscarnet, and cidofovir, and A543V had 10-fold reduced susceptibility to cidofovir. Viral growth assays showed G441S, A543V, F345L, and P800L impaired viral growth capacities compared with wild-type AD169 HCMV. Three-dimensional modeling predicted A543V and A928T phenotypes but not R512C, reinforcing the need for individual characterization of mutations by recombinant phenotyping. CONCLUSIONS: Extending mutation databases is crucial to optimize treatments and to improve the assessment of patients with resistant/refractory HCMV infection.

Flexible multiplex PCR to detect SARS-CoV-2, coronavirus OC43 and influenza A virus in nasopharyngeal swab samples.

INTRODUCTION: Quantitative reverse transcription PCR (RT-qPCR) is the leading tool to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given that it will almost certainly continue to coexist with other respiratory viruses in the coming years, our study aimed to design a multiplex PCR system not affected by supplier outages and with reduced cost compared to the existing commercially available kits. METHODS AND RESULTS: In this study, combinations of four primers/probe sets were used to construct a flexible RT-qPCR assay which is capable of discriminating between SARS-CoV-2 and the seasonal human coronavirus HCoV-OC43, or even influenza A virus. Additionally, the human RPP30 gene was used as an internal control. To demonstrate the robustness of the assay, it was applied to a collection of 150 clinical samples. The results showed 100% sensitivity and specificity compared to the automatized system used at the hospital and were better when indeterminate samples were analysed. CONCLUSIONS: This study provides an efficient method for the simultaneous detection of SARS-CoV-2, HCoV-OC43 and influenza A virus, and its efficacy has been tested on clinical samples showing outstanding results. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex RT-qPCR design offers an accessible and economical alternative to commercial detection kits for hospitals and laboratories with limited economic resources or facing situations of supply shortage.

Re-emergence of enterovirus D68 in Europe after easing the COVID-19 lockdown, September 2021.

We report a rapid increase in enterovirus D68 (EV-D68) infections, with 139 cases reported from eight European countries between 31 July and 14 October 2021. This upsurge is in line with the seasonality of EV-D68 and was presumably stimulated by the widespread reopening after COVID-19 lockdown. Most cases were identified in September, but more are to be expected in the coming months. Reinforcement of clinical awareness, diagnostic capacities and surveillance of EV-D68 is urgently needed in Europe.

Molecular epidemiology of an enterovirus A71 outbreak associated with severe neurological disease, Spain, 2016.

IntroductionEnterovirus A71 (EV-A71) is an emerging pathogen that causes a wide range of disorders including severe neurological manifestations. In the past 20 years, this virus has been associated with large outbreaks of hand, foot and mouth disease with neurological complications in the Asia-Pacific region, while in Europe mainly sporadic cases have been reported. In spring 2016, however, an EV-A71 outbreak associated with severe neurological cases was reported in Catalonia and spread further to other Spanish regions.AimOur objective was to investigate the epidemiology and clinical characteristics of the outbreak.MethodsWe carried out a retrospective study which included 233 EV-A71-positive samples collected during 2016 from hospitalised patients. We analysed the clinical manifestations associated with EV-A71 infections and performed phylogenetic analyses of the 3'-VP1 and 3Dpol regions from all Spanish strains and a set of EV-A71 from other countries.ResultsMost EV-A71 infections were reported in children (mean age: 2.6 years) and the highest incidence was between May and July 2016 (83%). Most isolates (218/233) were classified as subgenogroup C1 and 217 of them were grouped in one cluster phylogenetically related to a new recombinant variant strain associated with severe neurological diseases in Germany and France in 2015 and 2016. Moreover, we found a clear association of EV-A71-C1 infection with severe neurological disorders, brainstem encephalitis being the most commonly reported.ConclusionAn emerging recombinant variant of EV-A71-C1 was responsible for the large outbreak in 2016 in Spain that was associated with many severe neurological cases.

Immunosuppression for inflammatory bowel disease does not influence Epstein-Barr viral load in the short-term.

INTRODUCTION: Immunomodulators and biologics are two of the main drugs used for the treatment of inflammatory bowel disease (IBD). Some of these agents have been associated with certain infections and lymphoproliferative disorders, including Epstein-Barr virus (EBV) infection. Our aim was to determine the influence of immunosuppression in the EBV viral load in patients with IBD. MATERIALS AND METHODS: We prospectively included naïve patients with IBD who were starting immunosuppressive therapy in four IBD Units. All patients were assessed at baseline and four months after starting immunosuppression for clinical disease activity, biomarkers, EBV serology (IgM VCA, IgG VCA and IgG EBNA) and viral load. RESULTS: Thirty-two patients were included. At baseline, all patients showed positive results for IgG VCA or IgG EBNA with undetectable EBV viral load. No patient showed detectable EBV viral load after starting the immunosuppressive therapy. CONCLUSION: Immunosuppression did not influence on EBV viral load in the short-term in naïve IBD patients.

Repeated Emergency Department Visits Among Children with Invasive Bacterial Infections.

We carried out a retrospective cohort study of 271 previously healthy children younger than 14 years old diagnosed with invasive bacterial infection in an emergency department. Of them, 72 (26.6%) had previous visits to the emergency department. Not identifying children with an invasive bacterial infection and not administering antibiotics on the first visit was associated with a severe outcome.

Blood enterovirus polymerase chain reaction testing in young febrile infants.

OBJECTIVE: To analyse the impact of blood enterovirus and human parechovirus PCR (ev-PCR) testing in young infants with fever without a source (FWS). DESIGN: Observational study, subanalysis of a prospective registry. SETTING: Paediatric emergency department. PATIENTS: Infants ≤90 days of age with FWS seen between September 2015 and August 2019 with blood ev-PCR, blood and urine cultures and urine dipstick test performed. MAIN OUTCOME MEASURES: Prevalence of invasive bacterial infection (IBI: bacterial pathogen in blood or cerebrospinal fluid) in infants with positive or negative ev-PCR test results. Secondarily, we also compared length of stay and antibiotic treatment in hospitalised infants. RESULTS: Of 703 infants, 174 (24.7%) had a positive blood ev-PCR and none of them were diagnosed with an IBI (vs 2.6% (95% CI 1.3% to 4.0%) of those with a negative result, p=0.02). Prevalence of non-IBI (mainly urinary tract infection) was also lower among infants with a positive blood ev-PCR (2.3% (95% CI 0.1% to 4.5%) vs 17.6% (95% CI 14.3% to 20.8%), p<0.01).Overall, 258 infants were hospitalised (36.6%) and 193 (74.8%) of them received antibiotics. Length of hospital stay and antibiotic treatment were shorter in those with a positive blood ev-PCR (median: 3 days vs 5 days and 1 day vs 5 days, respectively; p<0.01). Differences remained statistically significant among well-appearing infants >21 days old with normal urine dipstick. CONCLUSION: Blood ev-PCR identifies a group of infants under 90 days of age with FWS at very low risk of IBI. This test may help to guide clinical decision making in young febrile infants.

Molecular characterization of multidrug resistant Enterobacterales strains isolated from liver and kidney transplant recipients in Spain.

The objective of this study was to analyse the mechanisms of resistance to carbapenems and other extended-spectrum-ß-lactams and to determine the genetic relatedness of multidrug-resistant Enterobacterales (MDR-E) causing colonization or infection in solid-organ transplantation (SOT) recipients. Prospective cohort study in kidney (n = 142), liver (n = 98) or kidney/pancreas (n = 7) transplant recipients between 2014 and 2018 in seven Spanish hospitals. We included 531 MDR-E isolates from rectal swabs obtained before transplantation and weekly for 4-6 weeks after the procedure and 10 MDR-E from clinical samples related to an infection. Overall, 46.2% Escherichia coli, 35.3% Klebsiella pneumoniae, 6.5% Enterobacter cloacae, 6.3% Citrobacter freundii and 5.7% other species were isolated. The number of patients with MDR-E colonization post-transplantation (176; 71.3%) was 2.5-fold the number of patients colonized pre-transplantation (71; 28.7%). Extended-spectrum ß-lactamases (ESBLs) and carbapenemases were detected in 78.0% and 21.1% of MDR-E isolates respectively. In nine of the 247 (3.6%) transplant patients, the microorganism causing an infection was the same strain previously cultured from surveillance rectal swabs. In our study we have observed a low rate of MDR-E infection in colonized patients 4-6 weeks post-transplantation. E. coli producing blaCTX-M-G1 and K. pneumoniae harbouring blaOXA-48 alone or with blaCTX-M-G1 were the most prevalent MDR-E colonization strains in SOT recipients.

Adherence to Human Colon Cells by Multidrug Resistant Enterobacterales Strains Isolated From Solid Organ Transplant Recipients With a Focus on Citrobacter freundii.

Enterobacteria species are common causes of hospital-acquired infections, which are associated with high morbidity and mortality rates. Immunocompromised patients such as solid organ transplant (SOT) recipients are especially at risk because they are frequently exposed to antibiotics in the course of their treatments. In this work, we used a collection of 106 Escherichia coli, 78 Klebsiella pneumoniae, 25 Enterobacter spp., and 24 Citrobacter spp. multidrug resistant strains isolated from transplant patients (hepatic, renal or renal/pancreatic) in order to examine their ability to adhere in vitro to HT-29 human colon cells, and to determine if some adhesive characteristics are associated with prevalence and persistence of these strains. A total of 33 E. coli (31%), 21 K. pneumoniae (27%), 7 Enterobacter spp. (28%), and 5 Citrobacter spp. (21%), adhered to the colon epithelial cells. Two main adherence patterns were observed in the four species analyzed, diffuse adherence, and aggregative adherence. Under transmission electronic microscopy (TEM), most bacteria lacked visible fimbria on their surface, despite their strong adherence to epithelial cells. None of the strains studied was able to induce any cytotoxic effect on HT-29 cells although some of them strongly colonizing both cells and glass coverslips at high density. Some of the strains failed to adhere to the epithelial cells but adhered strongly to the cover-slide, which shows that microscopy studies are mandatory to elucidate the adherence of bacteria to epithelial cells in vitro, and that quantitative assays using colony forming unit (CFUs) counting need to be supplemented with pictures to determine definitively if a bacterial strain adheres or not to animal cells in vitro. We report here, for the first time, the aggregative adherence pattern of two multidrug resistant (MDR) Citrobacter freundii strains isolated from human patients; importantly, biofilm formation in Citrobacter is totally dependent on the temperature; strong biofilms were formed at room temperature (RT) but not at 37°C, which can play an important role in the colonization of hospital surfaces. In conclusion, our results show that there is a great variety of adhesion phenotypes in multidrug-resistant strains that colonize transplanted patients.

Author Correction: Biofilm formation by multidrug resistant Enterobacteriaceae strains isolated from solid organ transplant recipients.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Enterovirus D68-associated respiratory and neurological illness in Spain, 2014-2018.

During 2014, enterovirus D68 (EV-D68) outbreaks were described globally, causing severe respiratory diseases in children and, in some cases, subsequent paralysis. In this study, the type characterization of enterovirus (EV) detected in respiratory illnesses and the epidemiology and clinical association of EV-D68 infections in Spain over a five-year period were described. A total of 546 EV-positive samples from hospitalized patients with respiratory infections were included. EV-D68 was the most frequently detected type (46.6%, 191/410 typed EV). Other EV from species A (25.1%), B (27.8%) and C (0.5%) were also identified. EV-D68 infections were more associated with bronchitis while EV-A/B types were more frequent in upper respiratory illness (p < 0.01). EV-D68 was also detected in patients with neurological symptoms (nine meningitis/meningoencephalitis and eight acute flaccid paralysis cases). Phylogenetic analysis of 3'-VP1 region showed most Spanish EV-D68 sequences from 2014 to 2016 belonged to subclades B2/B3, as other American and European strains circulating during the same period. However, those detected in 2017 and 2018 clustered to the emerged subclade D1. In summary, different EV can cause respiratory infections but EV-D68 was the most prevalent, with several strains circulating in Spain at least since 2014. Association between EV-D68 infection and neurological disease was also described.

Biofilm formation by multidrug resistant Enterobacteriaceae strains isolated from solid organ transplant recipients.

Solid organ transplant (SOT) recipients are especially at risk of developing infections by multidrug resistant bacteria (MDR). In this study, the biofilm-forming capability of 209 MDR strains (Escherichia coli n = 106, Klebsiella pneumoniae n = 78, and Enterobacter spp. n = 25) isolated from rectal swabs in the first 48 hours before or after kidney (93 patients), liver (60 patients) or kidney/pancreas transplants (5 patients) were evaluated by using a microplate assay. Thirty-nine strains were isolated before transplant and 170 strains were isolated post-transplant. Overall, 16% of E. coli strains, 73% of K. pneumoniae strains and 4% Enterobacter strains showed moderate or strong biofilm production. Nine strains isolated from infection sites after transplantation were responsible of infections in the first month. Of these, 4 K. pneumoniae, 1 E. coli and 1 Enterobacter spp. strains isolated pre-transplant or post-transplant as colonizers caused infections in the post-transplant period. Our results suggest that in vitro biofilm formation could be an important factor for adhesion to intestine and colonization in MDR K. pneumoniae strains in SOT recipients, but this factor appears to be less important for MDR E. coli and Enterobacter spp.

Improvement in detecting cytomegalovirus drug resistance mutations in solid organ transplant recipients with suspected resistance using next generation sequencing.

OBJETIVES: The aim of this study was to identify CMV drug resistance mutations (DRM) in solid organ transplant (SOT) recipients with suspected resistance comparing next-generation sequencing (NGS) with Sanger sequencing and assessing risk factors and the clinical impact of resistance. METHODS: Using Sanger sequencing as the reference method, we prospectively assessed the ability of NGS to detect CMV DRM in the UL97 and UL54 genes in a nationwide observational study from September 2013 to August 2016. RESULTS: Among 44 patients recruited, 14 DRM were detected by Sanger in 12 patients (27%) and 20 DRM were detected by NGS, in 16 (36%). NGS confirmed all the DRM detected by Sanger. The additional six mutations detected by NGS were present in <20% of the sequenced population, being located in the UL97 gene and conferring high-level resistance to ganciclovir. The presence of DRM by NGS was associated with lung transplantation (p = 0.050), the administration of prophylaxis (p = 0.039), a higher mean time between transplantation and suspicion of resistance (p = 0.038) and longer antiviral treatment duration before suspicion (p = 0.024). However, the latter was the only factor independently associated with the presence of DRM by NGS in the multivariate analysis (OR 2.24, 95% CI 1.03 to 4.87). CONCLUSIONS: NGS showed a higher yield than Sanger sequencing for detecting CMV resistance mutations in SOT recipients. The presence of DRM detected by NGS was independently associated with longer antiviral treatment.

Detection of cytomegalovirus drug resistance mutations in solid organ transplant recipients with suspected resistance.

BACKGROUND: Current guidelines recommend that treatment of resistant cytomegalovirus (CMV) in solid organ transplant (SOT) recipients must be based on genotypic analysis. However, this recommendation is not systematically followed. OBJECTIVES: To assess the presence of mutations associated with CMV resistance in SOT recipients with suspected resistance, their associated risk factors and the clinical impact of resistance. STUDY DESIGN: Using Sanger sequencing we prospectively assessed the presence of resistance mutations in a nation-wide prospective study between September 2013-August 2015. RESULTS: Of 39 patients studied, 9 (23%) showed resistance mutations. All had one mutation in the UL 97 gene and two also had one mutation in the UL54 gene. Resistance mutations were more frequent in lung transplant recipients (44% p=0.0068) and in patients receiving prophylaxis ≥6 months (57% vs. 17%, p=0.0180). The mean time between transplantation and suspicion of resistance was longer in patients with mutations (239 vs. 100days, respectively, p=0.0046) as was the median treatment duration before suspicion (45 vs. 16days, p=0.0081). There were no significant differences according to the treatment strategies or the mean CMV load at the time of suspicion. Of note, resistance-associated mutations appeared in one patient during CMV prophylaxis and also in a seropositive organ recipient. Incomplete suppression of CMV was more frequent in patients with confirmed resistance. CONCLUSIONS: Our study confirms the need to assess CMV resistance mutations in any patient with criteria of suspected clinical resistance. Early confirmation of the presence of resistance mutations is essential to optimize the management of these patients.