COVID-19-associated aspergillosis in a Brazilian referral centre: Diagnosis, risk factors and outcomes.

BACKGROUND: COVID-19 patients on mechanical ventilation are at risk to develop invasive aspergillosis. To provide additional data regarding this intriguing entity, we conducted a retrospective study describing risk factors, radiology and prognosis of this emerging entity in a Brazilian referral centre. METHODS: This retrospective study included intubated (≥18 years) patients with COVID-19 admitted from April 2020 until July 2021 that had bronchoscopy to investigate pulmonary co-infections. COVID-19-associated aspergillosis (CAPA) was defined according to the 2020 European Confederation of Medical Mycology/International Society of Human and Animal Mycosis consensus criteria. The performance of tracheal aspirate (TA) cultures to diagnose CAPA were described, as well as the radiological findings, risk factors and outcomes. RESULTS: Fourteen patients (14/87, 16%) had probable CAPA (0.9 cases per 100 ICU admissions). The sensitivity, specificity, positive predictive value and negative predictive value of TA for the diagnosis of CAPA were 85.7%, 73.1%, 46.2% and 95% respectively. Most of the radiological findings of CAPA were classified as typical of invasive pulmonary aspergillosis (64.3%). The overall mortality rate of probable CAPA was 71.4%. Age was the only independent risk factor for CAPA [p = .03; odds ratio (OR) 1.072]. CAPA patients under renal replacement therapy (RRT) may have a higher risk for a fatal outcome (p = .053, hazard ratio 8.047). CONCLUSIONS: CAPA was a prevalent co-infection in our cohort of patients under mechanical ventilation. Older patients had a higher risk to develop CAPA, and a poor prognosis may be associated with RRT.

Clostridioides difficile laboratory diagnostic techniques: a comparative approach of rapid and molecular methods.

Clostridioides difficile infection is a public health problem because of it is easily spread; with harmful consequences, it is essential to reduce hospital costs and prevent its dissemination by having a precise diagnosis. The gold standard for its diagnosis is polymerase chain reaction (PCR); however, the technique is not available for all laboratories due to the high cost. New approaches using non-molecular tests to detect C. difficile and toxin A/B production has been proposed to improve cost benefits. The objective of this study is to compare molecular methods (PCR) and rapid methods (immunochromatographic test and enzymatic immunoassay). A series of tests comprising these diagnostic techniques was performed with 50 patients with a clinical diagnosis for Clostridioides difficile on GeneXpert® devices test; a calculation of the sensitivity was executed, followed by a comparison of the efficiency of all techniques. Greater sensitivity was observed in the PCR-based methods (BD MAX™ and BioFire FilmArray®) and the GDH-based assays (RIDASCREEN® and Alere Techlab®). The proposed algorithm represents minor monetary disadvantages but a significant temporal optimization of 10%. Future studies concerning both positive and negative results could be advantageous because of the possibility of calculating more method concordance indexes, such as the specificity and Kappa index, in addition to being able to indicate a monetary profit if the proposed algorithm was applied due to the nonproceeding PCR cases.

Evaluation of Candida bloodstream infection and antifungal utilization in a tertiary care hospital.

BACKGROUND: Candida bloodstream infections carry a significant mortality risk, justifying the importance of adequate antifungal therapy. This study describes trends in antifungal consumption using the Defined Daily Dose (DDD) and Days of Therapy (DOT) metrics, identifies the microbiological profile, the time to initiation of empirical therapy, the adjustment after positive blood culture results for Candida, and the impact on in-hospital mortality rate in patients with candidemia. METHODS: An analysis of antifungal consumption from 2008 to 2016, and of candidemia cases from 2012 to 2016 was carried out in a private tertiary hospital. RESULTS: A total of 11,273 admissions were identified with a prescription for at least one type of antifungal therapy. Fluconazole was the most prescribed antifungal drug in terms of general consumption. Through the DDD and DOT metrics, we observed that over time, there was an increase in the consumption of liposomal amphotericin B, micafungin and voriconazole. Candida albicans was the most isolated species in blood cultures. Regarding candidemia, we analyzed samples from 115 patients. Empirical therapy was started within 24 h of blood culture in 44.3% of the cases, and in 81.7% of the cases, the antifungal was deemed to be adequate based in antifungal susceptibility testing, both of which were not associated with the in-hospital mortality rate. CONCLUSIONS: Our study reinforces the importance of monitoring the consumption of antifungal agents, which helps in proposing actions that lead to their rational use and, consequently, reduces the appearance of resistant strains.

Bacterial identification in herniated intervertebral discs: a prospective cohort study.

BACKGROUND CONTEXT: Reports of Cutibacterium acnes isolated in cultures of intervertebral disc samples suggest it as possibly responsible for inflammatory conditions causing Modic changes on spinal magnetic resonance imaging (MRI). PURPOSE: Our objective was to investigate the prevalence of C. acnes in samples of intervertebral disc of patients with lumbar disc herniation; to investigate prognostic factors and the relationship of Modic changes with infection 1 year after microdiscectomy. STUDY DESIGN: Prospective cohort study. PATIENT SAMPLE: In this single-center study, patients consecutively operated on for disc herniation had samples of the disc, multifidus muscle and ligamentum flavum (as an indication of contamination) extracted for culture. OUTCOME MEASURES: Age, sex, alcohol and tobacco consumption, body mass index; function, pain, and Modic chances in MRI before surgery and MRI 1 year later; rate of disc, muscle and ligament infection (primary outcome); diabetes and corticoid use (confoundings). METHODS: The protruded disc, muscle and ligament samples were sent for culture analysis in up to 30 minutes. A subsample of 17 patients underwent next-generation sequencing (NGS) molecular analysis too. We performed descriptive analysis and comparison of groups of patients with and without infection or contamination using Student's t, Mann-Whitney, chi-square, or Fisher's exact tests as appropriate, and pre- and postsurgical comparisons with the Wilcoxon test. RESULTS: From January 2018 to September 2019, 112 patients underwent open lumbar microdiscectomy, 67 (59.8%) men. Cultures showed 7 (6.3%) positive cases in the disc (2 with C. acnes), 3 (2.7%) in the ligament, and 12 (10, 7%) in muscle. No evidence of a difference in Modic alterations pre- or postoperatively was found between patients with and without positive culture 1 year after surgery. No association was found between culture positivity and functional or pain differences either. NGS results were all negative for C. acnes. CONCLUSIONS: We identified infective bacterial presence in the herniated disc in less than 2% of patients with disc herniation. C. acnes was not identified in any disc microbiome analysis. No significant association was observed between positivity for tissue infection and any clinical prognostic factor.

Evaluation of pathogen from the FilmArray meningitis/encephalitis panel and recommendations on atypical findings.

BACKGROUND: Infectious meningoencephalitis is a potentially fatal clinical condition that causes inflammation of the central nervous system secondary to the installation of different microorganisms. The FilmArray meningitis/encephalitis panel allows the simultaneous detection of 14 pathogens with results in about one hour. OBJECTIVE: This study is based on retrospectively evaluating the implementation of the FilmArray meningitis/encephalitis panel in a hospital environment, highlighting the general results and, especially, analyzing the consistency of the test results against the clinical and laboratory conditions of the patients. METHODS: Data were collected through the results reported by the BioFire FilmArray system software from the meningitis/encephalitis panel. The correlated laboratory tests used in our analysis, when available, included biochemical, cytological, direct and indirect microbiological tests. RESULTS: In the analyzed period, there were 496 samples with released results. Of the total of 496 samples analyzed, 88 (17.75%) were considered positive, and 90 pathogens were detected, and in 2 of these (2.27%) there was co-detection of pathogens. Viruses were the agents most frequently found within the total number of pathogens detected. Of the 496 proven samples, 20 (4.03%) were repeated, 5 of which were repeated due to invalid results, 6 due to the detection of multiple pathogens and 9 due to disagreement between the panel results and the other laboratory tests and/or divergence of the clinical-epidemiological picture. Of these 20 repeated samples, only 4 of them (20%) maintained the original result after repeating the test, with 16 (80%) being non-reproducible. The main factor related to the disagreement of these 16 samples during retesting was the detection of bacterial agents without any relationship with other laboratory tests or with the patients' clinical condition. CONCLUSION: In our study, simply reproducing tests with atypical results from the FilmArray meningitis/encephalitis panel proved, in most cases, effective and sufficient for interpreting these results.

ANTECEDENTES: A meningoencefalite infecciosa é uma condição clínica potencialmente fatal que causa inflamação do sistema nervoso central secundária à instalação de diversos microrganismos. O painel de meningite/encefalite FilmArray permite a detecção simultânea de 14 patógenos, com resultados em cerca de uma hora. OBJETIVO: Este estudo baseia-se em avaliar retrospectivamente a implementação do painel de meningite/encefalite FilmArray em ambiente hospitalar, destacando os resultados gerais e, principalmente, analisando a consistência dos resultados do teste frente às condições clínicas e laboratoriais dos pacientes. MéTODOS: Os dados foram coletados por meio dos resultados relatados pelo software do sistema BioFire FilmArray do painel de meningite/encefalite. Os exames laboratoriais correlacionados utilizados em nossa análise, quando disponíveis, incluíram exames bioquímicos, citológicos, microbiológicos diretos e indiretos. RESULTADOS: No período analisado, foram 496 amostras com resultados divulgados. Do total de 496 amostras analisadas, 88 (17,75%) foram consideradas positivas e 90 patógenos foram detectados, sendo que em duas destas (2,27%) houve codetecção de patógenos. Os vírus foram os agentes mais frequentemente encontrados dentro do total de patógenos detectados. Das 496 amostras analisadas, 20 (4,03%) foram repetidas, sendo 5 repetidas por resultado inválido, 6 pela detecção de múltiplos patógenos e 9 por discordância dos resultados do painel com os demais exames laboratoriais e/ou divergência do quadro clínico-epidemiológico. Destas 20 amostras repetidas, apenas 4 delas (20%) mantiveram o resultado original após a repetição do teste, sendo 16 (80%) não reprodutíveis. O principal fator relacionado à discordância destas 16 amostras na retestagem foi a detecção de agentes bacterianos sem qualquer relação com os demais exames laboratoriais ou com o quadro clínico dos pacientes. CONCLUSãO: Em nosso estudo, a simples repetição dos testes com resultados atípicos do painel de meningite/encefalite FilmArray mostrou-se, na maior dos casos, efetiva e suficiente para a interpretação destes achados.

Syphilis Treatment: Systematic Review and Meta-Analysis Investigating Nonpenicillin Therapeutic Strategies.

Background: Penicillin's long-standing role as the reference standard in syphilis treatment has led to global reliance. However, this dependence presents challenges, prompting the need for alternative strategies. We performed a systematic literature review and meta-analysis to evaluate the efficacy of these alternative treatments against nonneurological syphilis. Methods: We searched MEDLINE, the Cumulative Index to Nursing and Allied Health Literature, Embase, Cochrane, Scopus, and Web of Science from database inception to 28 August 2023, and we included studies that compared penicillin or amoxicillin monotherapy to other treatments for the management of nonneurological syphilis. Our primary outcome was serological cure rates. Random-effect models were used to obtain pooled mean differences, and heterogeneity was assessed using the I2 test. Results: Of 6478 screened studies, 27 met the inclusion criteria, summing 6710 patients. The studies were considerably homogeneous, and stratified analyses considering each alternative treatment separately revealed that penicillin monotherapy did not outperform ceftriaxone (pooled odds ratio, 1.66 [95% confidence interval, .97-2.84]; I2 = 0%), azithromycin (0.92; [.73-1.18]; I2 = 0%), or doxycycline (0.82 [.61-1.10]; I2 = 1%) monotherapies with respect to serological conversion. Conclusions: Alternative treatment strategies have serological cure rates equivalent to penicillin, potentially reducing global dependence on this antibiotic.

A hospital-based matched case-control study to identify clinical outcome and risk factors associated with carbapenem-resistant Klebsiella pneumoniae infection.

BACKGROUND: Healthcare-associated infections caused by Klebsiella pneumoniae isolates are increasing and few effective antibiotics are currently available to treat patients. We observed decreased carbapenem susceptibility among K. pneumoniae isolated from patients at a tertiary private hospital that showed a phenotype compatible with carbapenemase production although this group of enzymes was not detected in any sample. The aim of this study was to describe the epidemiology and clinical outcomes associated with carbapenem-resistant K. pneumoniae and to determine the antimicrobial resistance mechanisms. METHODS: Risk factors associated with carbapenem-resistant K. pneumoniae infections were investigated by a matched case-control study from January 2006 through August 2008. A cohort study was also performed to evaluate the association between carbapenem resistance and in-hospital mortality. Bacterial identification and antimicrobial susceptibility were determined by Vitek 2 and Etest. Carbapenemase activity was detected using spectrophotometric assays. Production of beta-lactamases and alterations in genes encoding K. pneumoniae outer membrane proteins, OmpK35 and OmpK36, were analyzed by PCR and DNA sequencing, as well as SDS-Page. Genetic relatedness of carbapenem resistant isolates was evaluated by Pulsed Field Gel Electrophoresis. RESULTS: Sixty patients were included (20 cases and 40 controls) in the study. Mortality was higher for patients with carbapenem-resistant K. pneumoniae infections compared with those with carbapenem-susceptible K. pneumoniae (50.0% vs 25.7%). The length of central venous catheter use was independently associated with carbapenem resistance in the multivariable analysis. All strains, except one, carried blaCTX-M-2, an extended-spectrum betalactamase gene. In addition, a single isolate also possessed blaGES-1. Genes encoding plasmid-mediated AmpC beta-lactamases or carbapenemases (KPC, metallo-betalactamases or OXA-carbapenemases) were not detected. CONCLUSIONS: The K. pneumoniae multidrug-resistant organisms were associated with significant mortality. The mechanisms associated with decreased K. pneumoniae carbapenem susceptibility were likely due to the presence of cephalosporinases coupled with porin alterations, which resulted from the presence of the insertion sequences in the outer membrane encoding genes.

Diagnostic Stewardship: A Systematic Review and Meta-analysis of Blood Collection Diversion Devices Used to Reduce Blood Culture Contamination and Improve the Accuracy of Diagnosis in Clinical Settings.

Background: Blood culture contamination may lead to misdiagnosis, overutilization of antibiotics, and prolonged length of stay. Blood specimen diversion devices can reduce contamination rates during blood culture collection procedures. We performed a systematic literature review and meta-analysis evaluating the influence of blood specimen diversion devices in blood culture contamination rates. Methods: We searched Medline, Cumulative Index to Nursing and Allied Health Literature, Embase, Cochrane, Scopus, and Web of Science, from database inception to 1 March 2023, for studies evaluating the impact of a diversion device on blood culture contamination. Blood culture contamination was a positive blood culture with microorganisms not representative of true bacteremia, but rather introduced during collection or processing the blood sample. Random-effects models were used to obtain pooled mean differences, and heterogeneity was assessed using the I2 test. Results: Of 1768 screened studies, 12 met inclusion criteria for this systematic literature review. Of them, 9 studies were included in the meta-analysis. Studies were substantially heterogeneous, but stratified analyses considering only high-quality studies revealed that venipuncture using a diversion device was associated with a significant reduction in blood culture contamination in comparison to the standard procedure of collection (pooled odds ratio [OR], 0.26 [95% confidence interval {CI}, .13-.54]; I2 = 19%). Furthermore, the stratified analysis showed that the adoption of a diversion device did not reduce the detection of true infection (pooled OR, 0.85 [95% CI, .65-1.11]; I2 = 0%). Conclusions: Blood culture diversion devices was associated with decreased contamination rates and could improve quality of care, reduce costs, and avoid unnecessary antibiotic use.

Characteristics, risk factors, and outcomes of bloodstream Candida infections in the intensive care unit: a retrospective cohort study.

OBJECTIVE: The main objective was to assess the clinical characteristics, associated factors, and outcomes of patients admitted to the ICU for candidemia. The secondary objective was to examine the relationship of candidemia with the length of stay and mortality. METHODS: The analysis was a retrospective single-center cohort study addressing the effect of invasive candidemia on outcomes. This study was performed in a medical-surgical ICU located in a tertiary private hospital in São Paulo, Brazil. Data was collected through the review of the hospital database. RESULTS: In total, 18,442 patients were included in our study, including 22 patients with candidemia. The median age was similar in patients with and without candidemia [67 (56-84) vs. 67 (51-80)]. Most patients were male, and the proportion of men was higher among patients with candidemia (77% vs. 55.3%). The rates of renal replacement therapy (40.9% vs. 3.3%), mechanical ventilation (63.6% vs. 29.6%), and parenteral nutrition (40.9% vs. 4.8%) were higher in patients with candidemia than in those without candidemia. The mortality rate (77.3% vs. 11.9%) and length of hospital stay [42 days (23.0-78.8) vs. 8 days (5.0-17.0)] were significantly higher in patients with candidemia. CONCLUSIONS: Patients with candidemia are prone to longer hospital stay and mortality. In addition, we found associations of candidemia with the use of invasive mechanical ventilation, renal replacement therapy, and parenteral nutrition.

Next-generation sequencing (NGS) to determine microbiome of herniated intervertebral disc.

BACKGROUND CONTEXT: There is apparent causality between chronic infection of the intervertebral disc and its degenerative process. Although disc is considered a sterile tissue, collected samples of uninfected patients sent to culture testing resulted positive. PURPOSE: The purpose of this study was to analyze the microbiome of the intervertebral disc by using and validating the next-generation sequencing (NGS) molecular test, controlled with tissue culture and clinical presentation of patients. STUDY DESIGN/SETTING: Prospective study of consecutive patients in a hospital. PATIENT SAMPLE: Patients with lumbar disc herniation undergoing open microdiscectomy aging 18 to 65 years. OUTCOME MEASURES: NGS, tissue culture METHODS: Subjects undergoing open decompression surgery for lumbar disc herniation were consecutively included and clinically followed for one year. Three samples of the excised herniated disc fragment were sent to tissue culture and another sample of the disc was sent to NGS test for microbiome analysis. Control samples of the ligamentum flavum and deep muscle were collected and sent to culture. RESULTS: A total of 17 patients were included. All patients presented negative cultures of the removed disc samples, as well as negative cultures of muscle and yellow ligament. None of the patients evolved to clinical infection one year after surgery, nor presented significant alteration of laboratory markers. NGS mapped a mean of 14,645 (range 6,540 to 27,176) DNA sequences for each disc sample of each patient. There were a total of 45 different bacteria genera remnants with different amount of DNA sequences detected. There was a mean of 8 (range 3-17) different bacterial elements in each sample of intervertebral disc. Three bacteria were present in all disc samples (Herbaspirillum, Ralstonia, and Burkolderia). Although there were a considerable mean number of bacterial sequences mapped in each disc sample, the amount of sequences related to bacteria was low. Cutibacterium acnes elements was not found in any disc microbiome analysis. CONCLUSIONS: NGS has been proven to adequately determine bacterial DNA presence within the intervertebral disc. C. acnes was not isolated in culture neither in microbiome analysis of patients with lumbar disc herniation. We cannot confirm disc sterility since, even if it does not cause infection, there is bacterial or remnant DNA in herniated discs.

The Gastrointestinal Load of Carbapenem-Resistant Enterobacteriacea Is Associated With the Transition From Colonization to Infection by Klebsiella pneumoniae Isolates Harboring the blaKPC Gene.

Background: Healthcare-associated infections by carbapenem-resistant Klebsiella pneumoniae are difficult to control. Virulence and antibiotic resistance genes contribute to infection, but the mechanisms associated with the transition from colonization to infection remain unclear. Objective: We investigated the transition from carriage to infection by K. pneumoniae isolates carrying the K. pneumoniae carbapenemase-encoding gene bla KPC (KpKPC). Methods: KpKPC isolates detected within a 10-year period in a single tertiary-care hospital were characterized by pulsed-field gel electrophoresis (PFGE), multilocus sequencing typing, capsular lipopolysaccharide and polysaccharide typing, antimicrobial susceptibility profiles, and the presence of virulence genes. The gastrointestinal load of carbapenem-resistant Enterobacteriaceae and of bla KPC-carrying bacteria was estimated by relative quantification in rectal swabs. Results were evaluated as contributors to the progression from carriage to infection. Results: No PGFE type; ST-, K-, or O-serotypes; antimicrobial susceptibility profiles; or the presence of virulence markers, such yersiniabactin and colibactin, were associated with carriage or infection, with ST437 and ST11 being the most prevalent clones. Admission to intensive and semi-intensive care units was a risk factor for the development of infections (OR 2.79, 95% CI 1.375 to 5.687, P=0.005), but higher intestinal loads of carbapenem-resistant Enterobacteriaceae or of bla KPC-carrying bacteria were the only factors associated with the transition from colonization to infection in this cohort (OR 8.601, 95% CI 2.44 to 30.352, P<0.001). Conclusion: The presence of resistance and virulence mechanisms were not associated with progression from colonization to infection, while intestinal colonization by carbapenem-resistant Enterobacteriacea and, more specifically, the load of gastrointestinal carriage emerged as an important determinant of infection.

Joint report of SBI (Brazilian Society of Infectious Diseases), FEBRASGO (Brazilian Federation of Gynecology and Obstetrics Associations), SBU (Brazilian Society of Urology) and SBPC/ML (Brazilian Society of Clinical Pathology/Laboratory Medicine): recommendations for the clinical management of lower urinary tract infections in pregnant and non-pregnant women.

Urinary tract infection (UTI) is a common condition in women. There is an increased concern on reduction of bacterial susceptibility resulting from wrongly prescribing antimicrobials. This paper summarizes the recommendations of four Brazilian medical societies (SBI - Brazilian Society of Infectious Diseases, FEBRASGO - Brazilian Federation of Gynecology and Obstetrics Associations, SBU - Brazilian Society of Urology, and SBPC/ML - Brazilian Society of Clinical Pathology/Laboratory Medicine) on the management of urinary tract infection in women. Asymptomatic bacteriuria should be screened at least twice during pregnancy (early and in the 3rd trimester). All cases of significant bacteriuria (≥105CFU/mL in middle stream sample) should be treated with antimicrobials considering safety and susceptibility profile. In women with typical symptoms of cystitis, dipsticks are not necessary for diagnosis. Urine cultures should be collected in pregnant women, recurrent UTI, atypical cases, and if there is suspicion of pyelonephritis. First line antimicrobials for cystitis are fosfomycin trometamol in a single dose and nitrofurantoin, 100mg every 6hours for five days. Second line drugs are cefuroxime or amoxicillin-clavulanate for seven days. During pregnancy, amoxicillin and other cephalosporins may be used, but with a higher chance of therapeutic failure. In recurrent UTI, all episodes should be confirmed by urine culture. Treatment should be initiated only after urine sampling and with the same regimens indicated for isolated episodes. Prophylaxis options of recurrent UTI are behavioral measures, non-antimicrobial and antimicrobial prophylaxis. Vaginal estrogens may be recommended for postmenopausal women. Other non-antimicrobial prophylaxis, including cranberry and immunoprophylaxis, have weak evidence supporting their use. Antimicrobial prophylaxis may be offered as a continuous or postcoital scheme. In pregnant women, options are cephalexin, 250-500mg and nitrofurantoin, 100mg (contraindicated after 37 weeks of pregnancy). Nonpregnant women may use fosfomycin trometamol, 3g every 10 days, or nitrofurantoin, 100mg (continuous or postcoital).

One year cross-sectional study in adult and neonatal intensive care units reveals the bacterial and antimicrobial resistance genes profiles in patients and hospital surfaces.

Several studies have shown the ubiquitous presence of bacteria in hospital surfaces, staff, and patients. Frequently, these bacteria are related to HAI (healthcare-associated infections) and carry antimicrobial resistance (AMR). These HAI-related bacteria contribute to a major public health issue by increasing patient morbidity and mortality during or after hospital stay. Bacterial high-throughput amplicon gene sequencing along with identification of AMR genes, as well as whole genome sequencing (WGS), are biotechnological tools that allow multiple-sample screening for a diversity of bacteria. In this paper, we used these methods to perform a one-year cross sectional profiling of bacteria and AMR genes in adult and neonatal intensive care units (ICU and NICU) in a Brazilian public, tertiary hospital. Our results showed high abundances of HAI-related bacteria such as S. epidermidis, S. aureus, K. pneumoniae, A. baumannii complex, E. coli, E. faecalis, and P. aeruginosa in patients and hospital surfaces. Most abundant AMR genes detected throughout ICU and NICU were mecA, blaCTX-M-1 group, blaSHV-like, and blaKPC-like. We found that NICU environment and patients were more widely contaminated with pathogenic bacteria than ICU. Patient samples, despite the higher bacterial load, have lower bacterial diversity than environmental samples in both units. Finally, we also identified contamination hotspots in the hospital environment showing constant frequencies of bacterial and AMR contamination throughout the year. Whole genome sequencing (WGS), 16S rRNA oligotypes, and AMR identification allowed a high-resolution characterization of the hospital microbiome profile.

Mutagenesis Induced by Sub-Lethal Doses of Ciprofloxacin: Genotypic and Phenotypic Differences Between the Pseudomonas aeruginosa Strain PA14 and Clinical Isolates.

Bacterial resistance is a severe threat to global public health. Exposure to sub-lethal concentrations has been considered a major driver of mutagenesis leading to antibiotic resistance in clinical settings. Ciprofloxacin is broadly used to treat infections caused by Pseudomonas aeruginosa, whereas increased mutagenesis induced by sub-lethal concentrations of ciprofloxacin has been reported for the reference strain, PAO1, in vitro. In this study we report increased mutagenesis induced by sub-lethal concentrations of ciprofloxacin for another reference strain, PA14-UCBPP, and lower mutagenesis for clinical isolates when compared to the reference strain. This unexpected result may be associated with missense mutations in imuB and recX, involved in adaptive responses, and the presence of Pyocin S2, which were found in all clinical isolates but not in the reference strain genome. The genetic differences between clinical isolates of P. aeruginosa and the reference PA14-UCBPP, often used to study P. aeruginosa phenotypes in vitro, may be involved in reduced mutagenesis under sub-lethal concentrations of CIP, a scenario that should be further explored for the understanding of bacterial fitness in hospital environments. Moreover, we highlight the presence of a complete umuDC operon in a P. aeruginosa clinical isolate. Even though the presence of umuDC did not contribute to a significant increase in mutagenesis, it highlights the dynamic exchange of genetic material between bacterial species in the hospital environment.

Evaluation of pathogen from the FilmArray meningitis/encephalitis panel and recommendations on atypical findings / Avaliação do patógeno do painel de meningite/encefalite FilmArray e recomendações sobre achados atípicos

Abstract Background Infectious meningoencephalitis is a potentially fatal clinical condition that causes inflammation of the central nervous system secondary to the installation of different microorganisms. The FilmArray meningitis/encephalitis panel allows the simultaneous detection of 14 pathogens with results in about one hour. Objective This study is based on retrospectively evaluating the implementation of the FilmArray meningitis/encephalitis panel in a hospital environment, highlighting the general results and, especially, analyzing the consistency of the test results against the clinical and laboratory conditions of the patients. Methods Data were collected through the results reported by the BioFire FilmArray system software from the meningitis/encephalitis panel. The correlated laboratory tests used in our analysis, when available, included biochemical, cytological, direct and indirect microbiological tests. Results In the analyzed period, there were 496 samples with released results. Of the total of 496 samples analyzed, 88 (17.75%) were considered positive, and 90 pathogens were detected, and in 2 of these (2.27%) there was co-detection of pathogens. Viruses were the agents most frequently found within the total number of pathogens detected. Of the 496 proven samples, 20 (4.03%) were repeated, 5 of which were repeated due to invalid results, 6 due to the detection of multiple pathogens and 9 due to disagreement between the panel results and the other laboratory tests and/or divergence of the clinical-epidemiological picture. Of these 20 repeated samples, only 4 of them (20%) maintained the original result after repeating the test, with 16 (80%) being non-reproducible. The main factor related to the disagreement of these 16 samples during retesting was the detection of bacterial agents without any relationship with other laboratory tests or with the patients' clinical condition. Conclusion In our study, simply reproducing tests with atypical results from the FilmArray meningitis/encephalitis panel proved, in most cases, effective and sufficient for interpreting these results.

Resumo Antecedentes A meningoencefalite infecciosa é uma condição clínica potencialmente fatal que causa inflamação do sistema nervoso central secundária à instalação de diversos microrganismos. O painel de meningite/encefalite FilmArray permite a detecção simultânea de 14 patógenos, com resultados em cerca de uma hora. Objetivo Este estudo baseia-se em avaliar retrospectivamente a implementação do painel de meningite/encefalite FilmArray em ambiente hospitalar, destacando os resultados gerais e, principalmente, analisando a consistência dos resultados do teste frente às condições clínicas e laboratoriais dos pacientes. Métodos Os dados foram coletados por meio dos resultados relatados pelo software do sistema BioFire FilmArray do painel de meningite/encefalite. Os exames laboratoriais correlacionados utilizados em nossa análise, quando disponíveis, incluíram exames bioquímicos, citológicos, microbiológicos diretos e indiretos. Resultados No período analisado, foram 496 amostras com resultados divulgados. Do total de 496 amostras analisadas, 88 (17,75%) foram consideradas positivas e 90 patógenos foram detectados, sendo que em duas destas (2,27%) houve codetecção de patógenos. Os vírus foram os agentes mais frequentemente encontrados dentro do total de patógenos detectados. Das 496 amostras analisadas, 20 (4,03%) foram repetidas, sendo 5 repetidas por resultado inválido, 6 pela detecção de múltiplos patógenos e 9 por discordância dos resultados do painel com os demais exames laboratoriais e/ou divergência do quadro clínico-epidemiológico. Destas 20 amostras repetidas, apenas 4 delas (20%) mantiveram o resultado original após a repetição do teste, sendo 16 (80%) não reprodutíveis. O principal fator relacionado à discordância destas 16 amostras na retestagem foi a detecção de agentes bacterianos sem qualquer relação com os demais exames laboratoriais ou com o quadro clínico dos pacientes. Conclusão Em nosso estudo, a simples repetição dos testes com resultados atípicos do painel de meningite/encefalite FilmArray mostrou-se, na maior dos casos, efetiva e suficiente para a interpretação destes achados.

[Staphylococcus cohnii spp urealyticus: case report on an uncommon pathogen]. / Staphylococcus cohnii spp urealyticus: relato de caso de um patógeno incomum.

Coagulase-negative Staphylococcus has emerged as an important agent in nosocomial infections. In this study, we report a case of bacteremia associated with a central venous catheter, caused by Staphylococcus cohnii spp urealyticus that was isolated in blood cultures from a 53-year-old male patient who was admitted to a general hospital in the city of São Paulo. We discuss in this report the difficulty in routinely identifying this microorganism in the clinical microbiology laboratory. Staphylococcus cohnii spp urealyticus is a microorganism found in human skin as part of the normal microbiota, and it can cause serious infections in humans, in some situations.

Evaluation of two commercial methods for the susceptibility testing of Candida species: Vitek 2® and Sensititre YeastOne®.

BACKGROUND: An increased incidence of fungal infections caused by Candida species, especially Candida glabrata and Candida krusei, which are less susceptible to azoles, has been observed. Standardized susceptibility testing is essential for clinical management and for monitoring the epidemiology of resistance. AIMS: We evaluated the performance of two different susceptibility testing commercial methods, Vitek 2® and Sensititre YeastOne®, and compared them with the standard broth microdilution method (CLSI). METHODS: A total of 80 isolates of several Candida species (Candida albicans, Candida parapsilosis complex, Candida tropicalis, C. glabrata and C. krusei) were selected for this study. RESULTS: We analyzed the categorical agreement (CA) between the methods, stratifying the disagreements. The average CA between the methods was 96.3% for Vitek 2® and 84% for Sensititre YeastOne®. No very major errors were observed. Major errors and minor errors were found for all the isolates tested. With the azoles, both Vitek 2® and Sensititre YeastOne® had good and similar performance levels, except for C. tropicalis and C. krusei (Sensititre YeastOne® showed low CA, 56.2%). With the echinocandins, both methods showed good performance for C. albicans, C. parapsilosis and C. tropicalis. However, we observed important discrepancies for C. krusei with caspofungin: Vitek 2® had 100% CA while Sensititre YeastOne® had only 25%. With amphotericin B, both Vitek 2® and Sensititre YeastOne® had good performance with high CA. CONCLUSIONS: Despite the limited isolates tested, we concluded that both methods have good performance and are reliable for antifungal susceptibility testing. However, caspofungin activity against C. krusei and C. glabrata should be interpreted carefully when using Sensititre YeastOne® because we observed a low CA.

Comparative Genomics of Nonoutbreak Pseudomonas aeruginosa Strains Underlines Genome Plasticity and Geographic Relatedness of the Global Clone ST235.

Pseudomonas aeruginosa is an important opportunistic pathogen in hospitals, responsible for various infections that are difficult to treat due to intrinsic and acquired antibiotic resistance. Here, 20 epidemiologically unrelated strains isolated from patients in a general hospital over a time period of two decades were analyzed using whole genome sequencing. The genomes were compared in order to assess the presence of a predominant clone or sequence type (ST). No clonal structure was identified, but core genome-based single nucleotide polymorphism (SNP) analysis distinguished two major, previously identified phylogenetic groups. Interestingly, most of the older strains isolated between 1994 and 1998 harbored exoU, encoding a cytotoxic phospholipase. In contrast, most strains isolated between 2011 and 2016 were exoU-negative and phylogenetically very distinct from the older strains, suggesting a population shift of nosocomial P. aeruginosa over time. Three out of 20 strains were ST235 strains, a global high-risk clonal lineage; these carried several additional resistance determinants including aac(6')Ib-cr encoding an aminoglycoside N-acetyltransferase that confers resistance to fluoroquinolones. Core genome comparison with ST235 strains from other parts of the world showed that the three strains clustered together with other Brazilian/Argentinean isolates. Despite this regional relatedness, the individuality of each of the three ST235 strains was revealed by core genome-based SNPs and the presence of genomic islands in the accessory genome. Similarly, strain-specific characteristics were detected for the remaining strains, indicative of individual evolutionary histories and elevated genome plasticity.

Laboratory detection methods for methicillin resistance in coagulase negative Staphylococcus isolated from ophthalmic infections.

PURPOSE: To evaluate different methods of oxacillin susceptibility testing of ocular isolates, considering polymerase chain reaction (PCR) as the 'gold standard', and to compare the in vitro susceptibility to oxacillin with that of other antimicrobials used in ophthalmologic practice. METHODS: The Vitek gram-positive identification card was used to identify ocular coagulase negative Staphylococcus species. The presence of the mecA gene was determined by the polymerase chain reaction assay with a combination of two primer sets (mecA and 16S rRNA) in a single region. Results were analyzed and compared with other oxacillin susceptibility methods: PBP2a detection by rapid slide latex agglutination test (SLA); oxacillin E-test; the Vitek automated gram-positive susceptibility card (GPS-105); the oxacillin salt agar screening test (OSAS) at a concentration of 6.0, 1.0 and 0.75 microg oxacillin per ml and the cefoxitin disk diffusion test (CDD). Automated susceptibility was also determined to other antimicrobial agents (fluoroquinolones, penicillin G, amoxicillin-ampicillin, cefazolin, ampicillin-sulbactam, erythromycin, clindamycin, gentamicin, tetracycline, trimethoprim-sulfamethoxazole, vancomycin and rifampin. RESULTS: Of the 69 CoNS isolates tested, 71% were mecA-positive and 29% mecA-negative. All methods tested had a statistically significant agreement with polymerase chain reaction. There was a tendency of positive polymerase chain reaction predomination among the S. epidermidis isolates in comparison to non-epidermidis isolates, although this was not statistically significant (78.3% vs. 56.5%; chi2= 2.54; P= 0.11). The oxacillin salt agar screening test (0.75 microg oxacillin/ml) showed the best performance, with 100% sensitivity and negative predictive value; 95% specificity and 98% positive predictive value. Using the E-test, the mecA-positive isolates were statistically significantly more resistant to ciprofloxacin, ofloxacin, gatifloxacin and moxifloxacin (P= 0.002; P= 0.008; P= 0.002 and P= 0.003, respectively). There was a statistically significant higher proportion of resistance of the coagulase negative Staphylococcus mecA-positives for: penicillin G, amoxicillin-ampicillin, cefazolin, ampicillin-sulbactam, erythromycin, clindamycin, gentamicin and tetracycline (P< or =0.05). All coagulase negative Staphylococcus species were susceptible to vancomycin and there was no statistically significant correlation between the mecA-positive isolates and resistance to trimethoprim-sulfamethoxazole or to rifampin. CONCLUSIONS: In the present study, we found that the E-test and the oxacillin salt agar screening test S (0.75 microg oxacillin per ml), when compared with polymerase chain reaction, were the most accurate currently available methods to phenotypically detect oxacillin resistance of coagulase negative Staphylococcus species. This study demonstrated that a good option for screening of ocular isolates for oxacillin resistance in the microbiology laboratory is the cefoxitin disk diffusion test and the automated Vitek system. We believe it is important to have available methods that accurately detect methicillin resistance of the less commonly encountered species, chiefly because of their increasing importance as opportunistic pathogens.

Identification of Clostridium difficile Asymptomatic Carriers in a Tertiary Care Hospital.

BACKGROUND: The diagnosis of Clostridium difficile infection (CDI) increases concern that asymptomatic carriers of toxigenic C. difficile may be diagnosed with CDI. METHODS: A matched case control study was conducted in inpatients in a tertiary care center. The first 50 patients with diarrhea and a positive polymerase chain reaction (PCR) test beginning February 1, 2015, were identified as cases. Control patients were hospitalized patients receiving antibiotics, but with no diarrhea, housed in a room as close as possible to each case during the same admission time. A convenience sample of healthcare workers who cared for C. difficile infected patients was also tested. RESULTS: We found two positive PCR results for C. difficile in controls (4.1%). None of these healthcare workers were positive for C. difficile by PCR. There was no difference between groups with respect to overall antibiotic use before the requested PCR for Clostridium difficile (p = 0.359). The majority of cases had a high proportion of gastrointestinal disorders (71.4%) compared with control (8.2%), p < 0.001. Patients with neoplasia had a higher chance of being identified as cases (p = 0.041). CONCLUSIONS: PCR should not be the only diagnostic tool but should be complementary to other methods and to the medical history.