Evaluation and optimization of the Sysmex UF1000i system for the screening of urinary tract infection in primary health care elderly patients

Objective Urinary tract infections (UTIs) are a common problem in the elderly population. Urine culture is still considered the "gold standard" to diagnose infection in this population. However, urine cultures are laborious and costly, and most samples will yield no growth. Methods An evaluation was made of the Sysmex UF-1000i flow cytometer as a screening tool for UTI in an elderly population older than 65 years who lived in the community, using 346 urine samples submitted for culture. Results The Receiver Operating Characteristic (ROC) analysis showed a significant difference (P < 0.01) between 0.98 bacteria area under the curve value and 0.82 of white blood cells (WBC). The combination of both counts for screening did not show any improvement in specificity or sensitivity. According to our data, the use of a single cut-off point of 200 bacteria/μL is suggested, in which the sensitivity and specificity were 99.11% and 91.59%, respectively, with a NPV of 99.49%. Moreover, this cut-off value could avoid 60.24% of the samples to be cultured, with a minimal false negative results rate of 0.87%. Conclusions The stratification of age groups stratification helps in selecting a more adjusted Sysmex UF1000i cut-off limit, leading to an improvement in the screening parameters that would imply a better management of these infections, as well as a high reduction in the workload and cost savings

Highly accurate whole-genome imputation of SARS-CoV-2 from partial or low-quality sequences

[Background] The current SARS-CoV-2 pandemic has emphasized the utility of viral whole-genome sequencing in the surveillance and control of the pathogen. An unprecedented ongoing global initiative is producing hundreds of thousands of sequences worldwide. However, the complex circumstances in which viruses are sequenced, along with the demand of urgent results, causes a high rate of incomplete and, therefore, useless sequences. Viral sequences evolve in the context of a complex phylogeny and different positions along the genome are in linkage disequilibrium. Therefore, an imputation method would be able to predict missing positions from the available sequencing data.[Results] We have developed the impuSARS application, which takes advantage of the enormous number of SARS-CoV-2 genomes available, using a reference panel containing 239,301 sequences, to produce missing data imputation in viral genomes. ImpuSARS was tested in a wide range of conditions (continuous fragments, amplicons or sparse individual positions missing), showing great fidelity when reconstructing the original sequences, recovering the lineage with a 100% precision for almost all the lineages, even in very poorly covered genomes (<20%).[Conclusions] Imputation can improve the pace of SARS-CoV-2 sequencing production by recovering many incomplete or low-quality sequences that would be otherwise discarded. ImpuSARS can be incorporated in any primary data processing pipeline for SARS-CoV-2 whole-genome sequencing.This work is supported by grant PT17/0009/0006 from the Spanish Ministry of Economy and Competitiveness, COVID-0012–2020 from Consejería de Salud y Familias, Junta de Andalucía, and postdoctoral contract PAIDI2020- DOC_00350 for C.L., from Junta de Andalucía, co-funded by the European Social Fund (FSE) 2014–2020.Peer reviewe

Molecular and phylogenetic characterization of the monkeypox outbreak in the South of Spain

Until the May 2022 Monkeypox outbreak, which spread rapidly to many non-endemic countries, the virus was considered a viral zoonosis limited to some African countries. The Andalusian circuit of genomic surveillance was rapidly applied to characterize the Monkeypox outbreak in the South of Spain. Whole genome sequencing was used to obtain the genomic profiles of samples collected across the south of Spain, representative of all the provinces of Andalusia. Phylogenetic analysis was used to study the relationship of the isolates and the available sequences of the 2022 outbreak. Whole genome sequencing of a total of 160 monkeypox viruses from the different provinces that reported cases were obtained. Interestingly, we report the sequences of monkeypox viruses obtained from two patients who died. While one of the isolates bore no noteworthy mutations that explain a potential heightened virulence, in another patient the second consecutive genome sequence, performed after the administration of tecovirimat, uncovered a mutation within the A0A7H0DN30 gene, known to be a prime target for tecovirimat in its Vaccinia counterpart. In general, a low number of mutations were observed in the sequences reported, which were very similar to the reference of the 2022 outbreak (OX044336), as expected from a DNA virus. The samples likely correspond to several introductions of the circulating monkeypox viruses from the last outbreak. The virus sequenced from one of the two patients that died presented a mutation in a gene that bears potential connections to drug resistance. This mutation was absent in the initial sequencing prior to treatmentThis work was supported by Spanish Ministry of Science and Innovation (grants PID2020- 117979RB-I00 and FJC2021-046546-I), the Instituto de Salud Carlos III (ISCIII), co-funded with European Regional Development Funds (ERDF) (grant IMP/00019), it has also been funded by Consejería de Salud y Consumo, Junta de Andalucía (grants COVID-0012-2020), and by grant ELIXIR-CONVERGE - Connect and align ELIXIR Nodes to deliver sustainable FAIR lifescience data management services (AMD-871075-16), funded by EU – H2020.N

Detection of High Level of Co-Infection and the Emergence of Novel SARS CoV-2 Delta-Omicron and Omicron-Omicron Recombinants in the Epidemiological Surveillance of Andalusia

Recombination is an evolutionary strategy to quickly acquire new viral properties inherited from the parental lineages. The systematic survey of the SARS-CoV-2 genome sequences of the Andalusian genomic surveillance strategy has allowed the detection of an unexpectedly high number of co-infections, which constitute the ideal scenario for the emergence of new recombinants. Whole genome sequence of SARS-CoV-2 has been carried out as part of the genomic surveillance programme. Sample sources included the main hospitals in the Andalusia region. In addition to the increase of co-infections and known recombinants, three novel SARS-CoV-2 delta-omicron and omicron-omicron recombinant variants with two break points have been detected. Our observations document an epidemiological scenario in which co-infection and recombination are detected more frequently. Finally, we describe a family case in which co-infection is followed by the detection of a recombinant made from the two co-infecting variants. This increased number of recombinants raises the risk of emergence of recombinant variants with increased transmissibility and pathogenicity.This research was funded by Spanish Ministry of Science and Innovation (grant PID2020-117979RB-I00), the Instituto de Salud Carlos III (ISCIII), co-funded with European Regional Development Funds (ERDF) (grant IMP/00019), and has also been funded by Consejería de Salud y Familias, Junta de Andalucía (grants COVID-0012-2020, PS-2020-342 and IE19_259 FPS).Peer reviewe

Dendritic cell deficiencies persist seven months after SARS-CoV-2 infection

Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)-2 infection induces an exacerbated inflammation driven by innate immunity components. Dendritic cells (DCs) play a key role in the defense against viral infections, for instance plasmacytoid DCs (pDCs), have the capacity to produce vast amounts of interferon-alpha (IFN-α). In COVID-19 there is a deficit in DC numbers and IFN-α production, which has been associated with disease severity. In this work, we described that in addition to the DC deficiency, several DC activation and homing markers were altered in acute COVID-19 patients, which were associated with multiple inflammatory markers. Remarkably, previously hospitalized and nonhospitalized patients remained with decreased numbers of CD1c+ myeloid DCs and pDCs seven months after SARS-CoV-2 infection. Moreover, the expression of DC markers such as CD86 and CD4 were only restored in previously nonhospitalized patients, while no restoration of integrin β7 and indoleamine 2,3-dyoxigenase (IDO) levels were observed. These findings contribute to a better understanding of the immunological sequelae of COVID-19

The imperative for quality control programs in Monkeypox virus DNA testing by PCR: CIBERINFEC quality control

© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.To evaluate molecular assays for Mpox diagnosis available in various clinical microbiology services in Spain through a quality control (QC) approach. A total of 14 centers from across Spain participated in the study. The Reference Laboratory dispatched eight serum samples and eight nucleic acid extracts to each participating center. Some samples were spiked with Mpox or Vaccinia virus to mimic positive samples for Mpox or other orthopox viruses. Participating centers provided information on the results obtained, as well as the laboratory methods used. Among the 14 participating centers seven different commercial assays were employed, with the most commonly used kit being LightMix Modular Orthopox/Monkeypox (Mpox) Virus (Roche®). Of the 12 centers conducting Mpox determinations, concordance ranged from 62.5% (n = 1) to 100% (n = 11) for eluates and from 75.0% (n = 1) to 100% (n = 10) for serum. Among the 10 centers performing Orthopoxvirus determinations, a 100% concordance was observed for eluates, while for serum, concordance ranged from 87.5% (n = 6) to 100% (n = 4). Repeatedly, 6 different centers reported a false negative in serum samples for Orthopoxvirus diagnosis, particularly in a sample with borderline Ct = 39. Conversely, one center, using the TaqMan™ Mpox Virus Microbe Detection Assay (Thermo Fisher), reported false positives in Mpox diagnosis for samples spiked with vaccinia virus due to cross-reactions. We observed a positive correlation of various diagnostic assays for Mpox used by the participating centers with the reference values. Our results highlight the significance of standardization, validation, and ongoing QC in the microbiological diagnosis of infectious diseases, which might be particularly relevant for emerging viruses.This research was supported by CIBER (Strategic Action for Monkeypox)—Consorcio Centro de Investigación Biomédica en Red—(CB 2021), Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación and Unión Europea—NextGenerationEU. A. d. S. is supported by ‘Instituto de Salud Carlos III’ (grant number JR22/00055).Peer reviewe

SARS-CoV-2 viral load in nasopharyngeal swabs is not an independent predictor of unfavorable outcome

The aim was to assess the ability of nasopharyngeal SARS-CoV-2 viral load at first patient’s hospital evaluation to predict unfavorable outcomes. We conducted a prospective cohort study including 321 adult patients with confirmed COVID-19 through RT-PCR in nasopharyngeal swabs. Quantitative Synthetic SARS-CoV-2 RNA cycle threshold values were used to calculate the viral load in log10 copies/mL. Disease severity at the end of follow up was categorized into mild, moderate, and severe. Primary endpoint was a composite of intensive care unit (ICU) admission and/or death (n = 85, 26.4%). Univariable and multivariable logistic regression analyses were performed. Nasopharyngeal SARS-CoV-2 viral load over the second quartile (≥ 7.35 log10 copies/mL, p = 0.003) and second tertile (≥ 8.27 log10 copies/mL, p = 0.01) were associated to unfavorable outcome in the unadjusted logistic regression analysis. However, in the final multivariable analysis, viral load was not independently associated with an unfavorable outcome. Five predictors were independently associated with increased odds of ICU admission and/or death: age ≥ 70 years, SpO2, neutrophils > 7.5 × 103/µL, lactate dehydrogenase ≥ 300 U/L, and C-reactive protein ≥ 100 mg/L. In summary, nasopharyngeal SARS-CoV-2 viral load on admission is generally high in patients with COVID-19, regardless of illness severity, but it cannot be used as an independent predictor of unfavorable clinical outcome

Assessing the Impact of SARS-CoV-2 Lineages and Mutations on Patient Survival

Objectives: More than two years into the COVID-19 pandemic, SARS-CoV-2 still remains a global public health problem. Successive waves of infection have produced new SARS-CoV-2 variants with new mutations for which the impact on COVID-19 severity and patient survival is uncertain. Methods: A total of 764 SARS-CoV-2 genomes, sequenced from COVID-19 patients, hospitalized from 19th February 2020 to 30 April 2021, along with their clinical data, were used for survival analysis. Results: A significant association of B.1.1.7, the alpha lineage, with patient mortality (log hazard ratio (LHR) = 0.51, C.I. = [0.14,0.88]) was found upon adjustment by all the covariates known to affect COVID-19 prognosis. Moreover, survival analysis of mutations in the SARS-CoV-2 genome revealed 27 of them were significantly associated with higher mortality of patients. Most of these mutations were located in the genes coding for the S, ORF8, and N proteins. Conclusions: This study illustrates how a combination of genomic and clinical data can provide solid evidence for the impact of viral lineage on patient survival.This work was supported by Spanish Ministry of Science and Innovation (grant PID2020- 117979RB-I00), the Instituto de Salud Carlos III (ISCIII), co-funded with European Regional Development Funds (ERDF) (grant IMP/00019), and has also been funded by Consejería de Salud y Familias, Junta de Andalucía (grants COVID-0012-2020 and PS-2020-342) and the postdoctoral contract of Carlos Loucera (PAIDI2020- DOC_00350), co-funded by the European Social Fund (FSE) 2014-2020. ELIXIR-CONVERGE—H2020 (871075).Peer reviewe

Effectiveness of Fosfomycin for the Treatment of Multidrug-Resistant Escherichia coli Bacteremic Urinary Tract Infections

IMPORTANCE The consumption of broad-spectrum drugs has increased as a consequence of the spread of multidrug-resistant (MDR) Escherichia coli. Finding alternatives for these infections is critical, for which some neglected drugs may be an option. OBJECTIVE To determine whether fosfomycin is noninferior to ceftriaxone or meropenem in the targeted treatment of bacteremic urinary tract infections (bUTIs) due to MDR E coli. DESIGN, SETTING, AND PARTICIPANTS This multicenter, randomized, pragmatic, open clinical trial was conducted at 22 Spanish hospitals from June 2014 to December 2018. Eligible participants were adult patients with bacteremic urinary tract infections due to MDR E coli; 161 of 1578 screened patients were randomized and followed up for 60 days. Data were analyzed in May 2021. INTERVENTIONS Patients were randomized 1 to 1 to receive intravenous fosfomycin disodium at 4 g every 6 hours (70 participants) or a comparator (ceftriaxone or meropenem if resistant; 73 participants) with the option to switch to oral fosfomycin trometamol for the fosfomycin group or an active oral drug or pa renteral ertapenem for the comparator group after 4 days. MAIN OUTCOMES AND MEASURES The primary outcome was clinical and microbiological cure (CMC) 5 to 7 days after finalization of treatment; a noninferiority margin of 7% was considered. RESULTS Among 143 patients in the modified intention-to-treat population (median [IQR] age, 72 [62-81] years; 73 [51.0%] women), 48 of 70 patients (68.6%) treated with fosfomycin and 57 of 73 patients (78.1%) treated with comparators reached CMC (risk difference, -9.4 percentage points; 1-sided 95% CI, -21.5 to infinity percentage points; P = .10). While clinical or microbiological failure occurred among 10 patients (14.3%) treated with fosfomycin and 14 patients (19.7%) treated with comparators (risk difference, -5.4 percentage points; 1-sided 95% CI. -infinity to 4.9; percentage points; P = .19), an increased rate of adverse event-related discontinuations occurred with fosfomycin vs comparators (6 discontinuations [8.5%] vs 0 discontinuations; P = .006). In an exploratory analysis among a subset of 38 patients who underwent rectal colonization studies, patients treated with fosfomycin acquired a new ceftriaxone-resistant or meropenem-resistant gram-negative bacteria at a decreased rate compared with patients treated with comparators (0 of 21 patients vs 4 of 17 patients [23.5%]; 1-sided P = .01). CONCLUSIONS AND RELEVANCE This study found that fosfomycin did not demonstrate noninferiority to comparators as targeted treatment of bUTI from MDR E coli; this was due to an increased rate of adverse event-related discontinuations. This finding suggests that fosfomycin may be considered for selected patients with these infections

Clinical and Ecological Impact of an Educational Program to Optimize Antibiotic Treatments in Nursing Homes (PROA-SENIOR): A Cluster, Randomized, Controlled Trial and Interrupted Time-Series Analysis

[Background] Antimicrobial stewardship programs (ASPs) are recommended in nursing homes (NHs), although data are limited. We aimed to determine the clinical and ecological impact of an ASP for NHs.[Methods] We performed a cluster, randomized, controlled trial and a before–after study with interrupted time-series analyses in 14 NHs for 30 consecutive months from July 2018 to December 2020 in Andalusia, Spain. Seven facilities implemented an ASP with a bundle of 5 educational measures (general ASP) and 7 added 1-to-1 educational interviews (experimental ASP). The primary outcome was the overall use of antimicrobials, calculated monthly as defined daily doses (DDD) per 1000 resident days (DRD).[Results] The total mean antimicrobial consumption decreased by 31.2% (−16.72 DRD; P = .045) with respect to the preintervention period; the overall use of quinolones and amoxicillin–clavulanic acid dropped by 52.2% (P = .001) and 42.5% (P = .006), respectively; and the overall prevalence of multidrug-resistant organisms (MDROs) decreased from 24.7% to 17.4% (P = .012). During the intervention period, 12.5 educational interviews per doctor were performed in the experimental ASP group; no differences were found in the total mean antimicrobial use between groups (−14.62 DRD; P = .25). Two unexpected coronavirus disease 2019 waves affected the centers increasing the overall mean use of antimicrobials by 40% (51.56 DRD; P < .0001).[Conclusions] This study suggests that an ASP for NHs appears to be associated with a decrease in total consumption of antimicrobials and prevalence of MDROs. This trial did not find benefits associated with educational interviews, probably due to the coronavirus disease 2019 pandemic.[Clinical Trials Registration] NCT03543605.Peer reviewe