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.

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.

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.

Performance of the dipstick screening test as a predictor of negative urine culture.

OBJECTIVE: To investigate whether the urine dipstick screening test can be used to predict urine culture results. METHODS: A retrospective study conducted between January and December 2014 based on data from 8,587 patients with a medical order for urine dipstick test, urine sediment analysis and urine culture. Sensitivity, specificity, positive and negative predictive values were determined and ROC curve analysis was performed. RESULTS: The percentage of positive cultures was 17.5%. Nitrite had 28% sensitivity and 99% specificity, with positive and negative predictive values of 89% and 87%, respectively. Leukocyte esterase had 79% sensitivity and 84% specificity, with positive and negative predictive values of 51% and 95%, respectively. The combination of positive nitrite or positive leukocyte esterase tests had 85% sensitivity and 84% specificity, with positive and negative predictive values of 53% and 96%, respectively. Positive urinary sediment (more than ten leukocytes per microliter) had 92% sensitivity and 71% specificity, with positive and negative predictive values of 40% and 98%, respectively. The combination of nitrite positive test and positive urinary sediment had 82% sensitivity and 99% specificity, with positive and negative predictive values of 91% and 98%, respectively. The combination of nitrite or leukocyte esterase positive tests and positive urinary sediment had the highest sensitivity (94%) and specificity (84%), with positive and negative predictive values of 58% and 99%, respectively. Based on ROC curve analysis, the best indicator of positive urine culture was the combination of positives leukocyte esterase or nitrite tests and positive urinary sediment, followed by positives leukocyte and nitrite tests, positive urinary sediment alone, positive leukocyte esterase test alone, positive nitrite test alone and finally association of positives nitrite and urinary sediment (AUC: 0.845, 0.844, 0.817, 0.814, 0.635 and 0.626, respectively). CONCLUSION: A negative urine culture can be predicted by negative dipstick test results. Therefore, this test may be a reliable predictor of negative urine culture. OBJETIVO: Verificar se a triagem de urina por fitas reativas é capaz de predizer a cultura de urina. Métodos Estudo retrospectivo realizado entre janeiro e dezembro de 2014 com 8.587 pacientes, com solicitação médica de triagem de urina (fita), sedimento urinário e cultura de urina. FORAM ANALISADOS: sensibilidade, especificidade, valor preditivo positivo, valor preditivo negativo e curva ROC. RESULTADOS: Foram positivas 17,5% das culturas. O nitrito apresentou sensibilidade de 28% e especificidade de 99%. O valor preditivo positivo foi de 89% e o valor preditivo negativo de 87%. Esterase apresentou sensibilidade de 79% e especificidade de 84%. Valor preditivo positivo e valor preditivo negativo foram de 51% e 95%, respectivamente. A combinação de nitrito ou esterase positivos apresentou sensibilidade de 85% e especificidade de 84%. Valor preditivo positivo e valor preditivo negativo foram, respectivamente, 53% e 96%. O sedimento positivo (mais de dez leucócitos por microlitro) apresentou sensibilidade de 92% e especificidade de 71%. O valor preditivo positivo foi 40% e o negativo, 98%. A combinação de nitrito e sedimento urinário positivos apresentou sensibilidade de 82% e especificidade de 99%. Os valores preditivos positivo e negativo foram 91% e 98%, respectivamente. Para o nitrito ou esterase positivos mais os leucócitos positivos, a sensibilidade foi de 94% e a especificidade de 84%. O valor preditivo positivo foi de 58% e o negativo foi de 99%. Com base na curva ROC, o melhor indicador de urocultura positiva foi a associação entre a esterase ou nitrito positivos na fita mais os leucócitos positivos no sedimento, seguido por nitrito e esterase positivos, sedimento urinário positivo isolado, esterase positiva isolada, nitrito positivo isolado e, finalmente, pela associação entre nitrito e sedimento urinário positivos (AUC: 0,845, 0,844, 0,817, 0,814, 0,635 e 0,626, respectivamente). CONCLUSÃO: Uma urocultura negativa pode ser prevista com resultados negativos na fita. Portanto, este teste pode ser um preditor confiável de urocultura negativa.

Evaluation of Two Methods for Determination of CD64 as a Diagnostic Marker of Infection in Critically Ill Adults.

Objectives. Diagnostic markers of infection have had little innovation over the last few decades. CD64, a marker expressed on the surface of neutrophils, may have utility for this purpose. Methods. This study was conducted in an adult intensive care unit (ICU) in São Paulo, Brazil, with 89 patients. We evaluated CD64 in patients with documented or clinically diagnosed infection (infection group) and controls (patients without any evidence of infection) by two different methodologies: method #1, an in house assay, and method #2, the commercial kit Leuko64 (Trillium Diagnostics). Results. CD64 displayed good discriminating power with a 91.2% sensitivity (95% CI 90.7-91.6%) for detecting infection. The commercial kit (Leuko64) demonstrated higher specificity (87.3%) compared with method #1 as well as better accuracy (88.8%). Conclusions. CD64 seems to be a promising marker of infection in the intensive care setting, with Leuko64 showing a slight advantage.

A multicenter trial of the efficacy and safety of tigecycline versus imipenem/cilastatin in patients with complicated intra-abdominal infections [Study ID Numbers: 3074A1-301-WW; ClinicalTrials.gov Identifier: NCT00081744].

BACKGROUND: Complicated intra-abdominal infections (cIAI) remain challenging to treat because of their polymicrobial etiology including multi-drug resistant bacteria. The efficacy and safety of tigecycline, an expanded broad-spectrum glycylcycline antibiotic, was compared with imipenem/cilastatin (IMI/CIS) in patients with cIAI. METHODS: A prospective, double-blind, multinational trial was conducted in which patients with cIAI randomly received intravenous (IV) tigecycline (100 mg initial dose, then 50 mg every 12 hours [q12h]) or IV IMI/CIS (500/500 mg q6h or adjusted for renal dysfunction) for 5 to14 days. Clinical response at the test-of-cure (TOC) visit (14-35 days after therapy) for microbiologically evaluable (ME) and microbiological modified intent-to-treat (m-mITT) populations were the co-primary efficacy endpoint populations. RESULTS: A total of 825 patients received >or= 1 dose of study drug. The primary diagnoses for the ME group were complicated appendicitis (59%), and intestinal (8.8%) and gastric/duodenal perforations (4.6%). For the ME group, clinical cure rates at TOC were 80.6% (199/247) for tigecycline versus 82.4% (210/255) for IMI/CIS (95% CI -8.4, 5.1 for non-inferiority tigecycline versus IMI/CIS). Corresponding clinical cure rates within the m-mITT population were 73.5% (227/309) for tigecycline versus 78.2% (244/312) for IMI/CIS (95% CI -11.0, 2.5). Nausea (31.0% tigecycline, 24.8% IMI/CIS [P = 0.052]), vomiting (25.7% tigecycline, 19.4% IMI/CIS [P = 0.037]), and diarrhea (21.3% tigecycline, 18.9% IMI/CIS [P = 0.435]) were the most frequently reported adverse events. CONCLUSION: This study demonstrates that tigecycline is as efficacious as imipenem/cilastatin in the treatment of patients with cIAI.

Robot-assisted needle insertion for venous catheterization.

Vein access can be challenging for a variety of patients. The development of robots-assisted central or peripheral veins puncture would facilitate life of health professionals and patients. New robots are under development for this purpose and probably they will become available for practical use in the near future. These techniques may decrease significantly the cost of medicine, which currently uses less informatics resources than other industries.

Fecal microbiota transplant by push enteroscopy to treat diarrhea caused by Clostridium difficile.

Clostridium difficile is the major etiological agent of pseudomembranous colitis and is found in up to 20% of adult inpatients. The recommended treatment is antibiotic therapy with metronidazole and/or vancomycin. However, the recurrence rate may reach up to 25% and it increases in each episode. The newest alternative to treat diarrhea due to recurrent Clostridium difficile is fecal microbiota transplantation. The procedure was performed in 12 patients, with a 6-month follow-up on 10 of them. Of the ten cases, bacterial recurrence was diagnosed in only one patient, after a course of antibiotic to treat urinary tract infection, without presenting with diarrhea. The particularity of our study, besides being an unprecedented event in South America, is the way to perform the infusion of fecal microbiota by enteroscopy.

Correlation between mass and volume of collected blood with positivity of blood cultures.

BACKGROUND: The collection of blood cultures is an extremely important method in the management of patients with suspected infection. Microbiology laboratories should monitor blood culture collection. METHODS: Over an 8-month period we developed a prospective, observational study in an adult Intensive Care Unit (ICU). We correlated the mass contained in the blood vials with blood culture positivity and we also verified the relationship between the mass of blood and blood volume collected for the diagnosis of bloodstream infection (BSI), as well as we explored factors predicting positive blood cultures. RESULTS: We evaluated 345 patients with sepsis, severe sepsis or septic shock for whom blood culture bottles were collected for the diagnosis of BSI. Of the 55 patients with BSI, 40.0% had peripheral blood culture collection only. BSIs were classified as nosocomial in 34.5%. In the multivariate model, the blood culture mass (in grams) remained a significant predictor of positivity, with an odds ratio 1.01 (i.e., for each additional 1 mL of blood collected there was a 1% increase in positivity; 95% CI 1.01-1.02, p = 0.001; Nagelkerke R Square [R(2)] = 0.192). For blood volume collected, the adjusted odds ratio was estimated at 1.02 (95% CI: 1.01-1.03, p < 0.001; R(2) = 0.199). For each set of collected blood cultures beyond one set, the adjusted odds ratio was estimated to be 1.27 (95% CI: 1.14-1.41, p < 0.001; R(2) = 0.221). CONCLUSIONS: Our study was a quality improvement project that showed that microbiology laboratories can use the weight of blood culture bottles to determine if appropriate volume has been collected to improve the diagnosis of BSI.

Evaluation of a new rapid test for carbapenemase detection in carbapenem resistant Enterobacteriaceae.

We evaluated a new phenotypic test for carbapenemase detection. A total of 100 Enterobacteriaceae isolates were selected. The test was compared with conventional PCR for bla(KPC) and bla(NDM) detection. We found 100% sensitivity and specificity, suggesting that this test may be a feasible alternative for rapid carbapenemase detection.

Characterizing uncommon Burkholderia cepacia complex isolates from an outbreak in a haemodialysis unit.

An outbreak of bacteraemia in a haemodialysis unit where 65 episodes of infection involved 35 outpatients is reported. Burkholderia cepacia complex was the agent most frequently recovered from blood. Thirty-three environmental and clinical isolates of B. cepacia complex were characterized by whole-cell protein electrophoresis and recA-RFLP profile. Fourteen isolates were genomovar I and 16 isolates were not classifiable by their recA-RFLP pattern. Ribotyping, random amplification of polymorphic DNA (RAPD) and integron profile were used to explore the clonality of the isolates, and revealed multiple strain genotypes. Four ribotypes and RAPD types and three integron patterns were identified. The water supply was identified as the source of the outbreak, and inappropriate cleaning and a leak in the reverse osmosis tubing connection were the probable causes of contamination. B. cepacia complex was still recovered from blood of patients even after apparently adequate measures were taken and water quality standards were met, suggesting that higher standards for water quality should be adopted in haemodialysis units. The genomovars recovered here were distinct from those commonly reported for cystic fibrosis isolates.

Reliability of hsp65-RFLP analysis for identification of Mycobacterium species in cultured strains and clinical specimens.

The purpose of this study was to evaluate the reliability of an amplification restriction analysis based method (hsp65-RFLP) to detect and identify mycobacterial species in clinical samples and cultures with low number of bacilli. We examined 247 clinical specimens and 88 culture vials, comparing hsp65-RFLP results with conventional culture/biochemical tests. The analytical sensitivity of the method was assessed with cerebrospinal fluid (CSF), broncho-alveolar lavage (BAL), sputum, water, and 12B medium containing defined amounts of mycobacterial chromosome. We detected the equivalent of 10(3) cells per ml in all samples, except sputum, the most common source of clinical sample for mycobacterial testing, which presented inhibition throughout. We investigated two purification procedures to overcome inhibition of DNA amplification: DNAzol and phenol/chloroform. The former was superior, eliminating inhibition in 93.7% of the clinical samples. The technique was effective for bacterial cultures, including those with very low growth indices (GIs), substantially abbreviating time for diagnosis, but showed low sensitivity (25%) when applied to clinical samples, an issue that has never been extensively assessed by other researchers.

Ventilator associated pneumonia: comparison between quantitative and qualitative cultures of tracheal aspirates.

INTRODUCTION: Deferred or inappropriate antibiotic treatment in ventilator-associated pneumonia (VAP) is associated with increased mortality, and clinical and radiological criteria are frequently employed to establish an early diagnosis. Culture results are used to confirm the clinical diagnosis and to adjust or sometimes withdraw antibiotic treatment. Tracheal aspirates have been shown to be useful for these purposes. Nonetheless, little is known about the usefulness of quantitative findings in tracheal secretions for diagnosing VAP. METHODS: To determine the value of quantification of bacterial colonies in tracheal aspirates for diagnosing VAP, we conducted a prospective follow-up study of 106 intensive care unit patients who were under ventilatory support. In total, the findings from 219 sequential weekly evaluations for VAP were examined. Clinical and radiological parameters were recorded and evaluated by three independent experts; a diagnosis of VAP required the agreement of at least two of the three experts. At the same time, cultures of tracheal aspirates were analyzed qualitatively and quantitatively (10(5) colony-forming units [cfu]/ml and 10(6) cfu/ml) RESULTS: Quantitative cultures of tracheal aspirates (10(5) cfu/ml and 10(6) cfu/ml) exhibited increased specificity (48% and 78%, respectively) over qualitative cultures (23%), but decreased sensitivity (26% and 65%, respectively) as compared with the qualitative findings (81%). Quantification did not improve the ability to predict a diagnosis of VAP. CONCLUSION: Quantitative cultures of tracheal aspirates in selected critically ill patients have decreased sensitivity when compared with qualitative results, and they should not replace the latter to confirm a clinical diagnosis of VAP or to adjust antimicrobial therapy.

Cardiac stem cells and their clinical use.

Stem cells have been used to correct the ravages of atherosclerotic heart disease and other diseases that involve acute deficit of myocardial muscle, such as myocarditis. Previous attempts at using bone marrow derived from stem cells have not been particularly successful. New technologies using cardiac stem cells seem to offer a better perspective of obtaining such regeneration.

Phylogenetic analysis of Stenotrophomonas spp. isolates contributes to the identification of nosocomial and community-acquired infections.

Stenotrophomonas ssp. has a wide environmental distribution and is also found as an opportunistic pathogen, causing nosocomial or community-acquired infections. One species, S. maltophilia, presents multidrug resistance and has been associated with serious infections in pediatric and immunocompromised patients. Therefore, it is relevant to conduct resistance profile and phylogenetic studies in clinical isolates for identifying infection origins and isolates with augmented pathogenic potential. Here, multilocus sequence typing was performed for phylogenetic analysis of nosocomial isolates of Stenotrophomonas spp. and, environmental and clinical strains of S. maltophilia. Biochemical and multidrug resistance profiles of nosocomial and clinical strains were determined. The inferred phylogenetic profile showed high clonal variability, what correlates with the adaptability process of Stenotrophomonas to different habitats. Two clinical isolates subgroups of S. maltophilia sharing high phylogenetic homogeneity presented intergroup recombination, thus indicating the high permittivity to horizontal gene transfer, a mechanism involved in the acquisition of antibiotic resistance and expression of virulence factors. For most of the clinical strains, phylogenetic inference was made using only partial ppsA gene sequence. Therefore, the sequencing of just one specific fragment of this gene would allow, in many cases, determining whether the infection with S. maltophilia was nosocomial or community-acquired.