RESUMO

PURPOSE: To evaluate the performance of the rapid colorimetric polymyxin B microelution (RCPEm) in determining polymyxin B resistance directly from Enterobacterales-positive blood cultures. METHODS: A set volume of positive blood culture bottles (diluted 1:10) was inoculated into a glucose-broth-phenol red solution (NP solution), where a polymyxin B disk was previously eluted (final concentration of 3 µg/mL). Test was read each 1 h for up to 4 h. Color change from red/orange to yellow indicated resistant isolates. Results were compared to the reference method, broth microdilution (BMD), performed from colonies grown on solid media from the same blood culture bottle. RESULTS: One hundred fifty-two Enterobacterales-positive blood cultures were evaluated, 22.4% (34/152) of them resistant to polymyxin B (including 6.6% with borderline MICs). When performing directly from positive blood cultures (RCPEm-BC), specificity and sensitivity were 99.1% and 94.1%, respectively. Of note, 79.4% (27/34) of truly resistant isolates required 3 h of incubation, compared to the 18 ± 2 h incubation that microtiter plates of BMD demand before reading can be performed. CONCLUSIONS: RCPEm directly from blood cultures has great potential to be part of the routine of clinical microbiology laboratories to establish polymyxin B susceptibility, impacting outcome of patients with bloodstream infections caused by carbapenem-resistant Enterobacterales.

Assuntos

RESUMO

The purpose of this study was to evaluate the MBT-ASTRA to determine susceptibility to ceftazidime/avibactam (CZA) and meropenem (MEM) of Enterobacterales directly from positive blood cultures (BC). Bacterial suspension was incubated with antibiotic and analyzed by MALDI-TOF MS. The relative growth was calculated and cutoff values were determined to categorize isolates as "S," "I," and "R." Klebsiella spp. with CZA 20/8 mg/L and 1.5-h incubation presented 1 (5.9%) major discrepancy and 96.3% category agreement; other species required 2.5 h for 100% category agreement. For MEM, 4 mg/L and 1.5h were necessary, demonstrating 2 (6.67%) minor discrepancies and 93.3% categorical agreement.

Assuntos

RESUMO

BACKGROUND: Fast and accurate detection of polymyxins resistance is necessary as they remain the last resources to treat infections caused by Carbapenem-resistant Enterobacterales in many regions. We evaluated the rapid colorimetric polymyxin B elution (RCPE) and developed its miniaturized version, RCPE microelution (RCPEm), aiming to detect polymyxins resistance among Enterobacterales. METHODS: The methodologies consist of exposing the bacterial population in a solution (NP solution) where polymyxin B disks were previously eluted to obtain a concentration of 2 µg/mL for RCPE and 3 µg/mL for RCPEm. RESULTS: Two hundred sixty-seven Enterobacterales were evaluated, 90 (33.7%) resistant to polymyxin B by broth microdilution. It was observed 0.6% of major error (ME) by RCPE, with a specificity of 99.4%. The miniaturized version (RCPEm) presented the same ME and specificity values, but slightly higher sensitivity (97.8% vs. 95.6%) with 2.2% of very major error (VME). CONCLUSIONS: RCPE and RCPEm proved to be useful alternatives to determine polymyxin B susceptibility in clinical microbiology laboratories, presenting low cost, being easy to perform, and demanding short incubation time.

Assuntos

RESUMO

Detecting carbapenemase-associated carbapenem resistance is a subject of major clinical and epidemiological concern as it influences therapeutic choice. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been proposed as a means to assess bacterial resistance mechanisms. We aimed to detect the KPC enzyme directly from positive blood cultures using MALDI-TOF MS. To do so, 102 clinical Enterobacteria were evaluated, including 59 blaKPC positives. Proteins were extracted using formic acid, isopropyl alcohol, and water (17:33:50) and spotted onto a steel target plate using the double-layer sinapinic acid technique. Two parameters were considered: (i) the visual detection of a clear peak with the expected KPC m/z and (ii) the evaluation of the relative intensity of the ions in the peak. A peak was observed in 56/59 blaKPC-positive isolates (94.9% sensitivity), with no false-positive results (100% specificity). When considering intensity, with a cut-off ≥120 (a.u.), sensitivity was 94.9% and specificity was 95.3%. We proposed a "buffer" zone, with intermediate values of intensity (115 to 125) reaching 100% sensitivity and specificity. The detection of KPC peaks directly from positive blood cultures using MALDI-TOF MS is feasible and rapid, which may improve appropriate patient therapy and antimicrobial stewardship.

RESUMO

In this study, we evaluate a method for the KPC enzyme detection, using MALDI-TOF MS, for Enterobacterales. A total of 300 clinical Enterobacterales isolates were selected. The collection included 259 carbapenemase-producing (157 KPC and 102 non-KPC) and 41 carbapenemase non-producing isolates. Bacterial proteins were extracted from Mueller-Hinton agar plates using formic acid, isopropyl alcohol, and water (17:33:50). Samples were prepared with a double layer of synapinic acid. Analyses were performed using a Microflex LT mass spectrometer (Bruker Daltonics) and flexAnalysis 4.0 software (Bruker Daltonics). Statistical analyses were performed using SPSS Software. A distinctive peak at m/z 28,643-28,731 was found in all 157 KPC-producing isolates, and it was consistently absent in the 143 KPC non-producing group. KPC-producing peak intensities ranged from 77 to 3893. Considering an intensity cutoff value ≥ 120 for the presence of KPC, this methodology presented 98.09% and 97.90% of sensitivity and specificity, respectively.

Assuntos

RESUMO

OBJECTIVES: To investigate the pattern of multiple human papillomavirus (HPV) infections and associated factors in young women who access the Brazilian public health care system to better understand the characteristics of multiple HPV infections, a critical issue in this era of multivalent vaccines. METHODS: This was a cross-sectional, multicenter study with sexually active unvaccinated women (16-25 years old) from 119 primary Brazilian healthcare centers between September 2016 and November 2017. Cervical samples were collected by trained health professionals, and HPV detection was performed in a central laboratory by Linear Array. RESULTS: Of the 5268 women, 33.00% (95% CI 31.07-34.92) had multiple infections. At least one type of high-risk HPV was present in 85.50% of all multiple infections. All HPV types were detected more frequently in association with other types than alone. Young individuals who were single or in a casual relationship and those who had more than one sexual partner in the past year were more likely to have multiple infections. CONCLUSIONS: In this work, a high rate of multiple HPV infections among unvaccinated young adults tended to increase due to certain risk factors. Such data can provide insight for decision makers in the development of public policies regarding HPV prevention.

Understanding the characteristics of multiple infections is critical in the era of HPV multivalent vaccines for the prevention of cervical carcinomas. Therefore, in this cross-sectional study, we aimed to investigate the pattern of multiple HPV infections and associated factors in 5,268 sexually active unvaccinated women (16­25 years old) who access the Brazilian public health care system. Cervical samples were collected by trained health professionals, and HPV detection was performed in a central laboratory by Linear Array. A total of 33.00% (95% CI 31.07­34.92) had multiple infections (60.43% of the HPV-positive sample). The number of HPV types in a multiple infection ranged from 2 to 14 different types. The viral types more frequently identified were HPV 16 and 52. All HPV types were detected more frequently in association with other types than alone. The incidence of multiple infections was 1.29 times higher in single than in married or cohabitating participants. Women who had two or more partners in the last year also had higher rates of multiple infections than those who had fewer than two sexual partners. In conclusion, a high prevalence of multiple infections prior to the national HPV immunization program was observed, especially with the increase in less safe behavior factors.

Assuntos

RESUMO

Recently, new combinations of ß-lactams and ß-lactamase inhibitors became available, including ceftazidime-avibactam, and increased the ability to treat infections caused by carbapenem-resistant Enterobacterales (CRE). Despite the reduced time of clinical use, isolates expressing resistance to ceftazidime-avibactam have been reported, even during treatment or in patients with no previous contact with this drug. Here, we detailed review data on global ceftazidime-avibactam susceptibility, the mechanisms involved in resistance, and the molecular epidemiology of resistant isolates. Ceftazidime-avibactam susceptibility remains high (≥ 98.4%) among Enterobacterales worldwide, being lower among extended-spectrum ß-lactamase (ESBL) producers and CRE. Alterations in class A ß-lactamases are the major mechanism involved in ceftazidime-avibactam resistance, and mutations are mainly, but not exclusively, located in the Ω loop of these enzymes. Modifications in Klebsiella pneumoniae carbapenemase (KPC) 3 and KPC-2 have been observed by many authors, generating variants with different mutations, insertions, and/or deletions. Among these, the most commonly described is Asp179Tyr, both in KPC-3 (KPC-31 variant) and in KPC-2 (KPC-33 variant). Changes in membrane permeability and overexpression of efflux systems may also be associated with ceftazidime-avibactam resistance. Although several clones have been reported, ST258 with Asp179Tyr deserves special attention. Surveillance studies and rationale use are essential to retaining the activity of this and other antimicrobials against class A CRE.

Assuntos

RESUMO

Detection of polymyxins susceptibility is challenging. We aimed to evaluate Rapid Polymyxin NP from colonies (NP-colony) and directly from positive blood bottles (NP-bottle), using polymyxin B instead of colistin among Enterobacterales. Both had similar and acceptable accuracy. This is the first study performing NP-bottle using polymyxin B instead of colistin.

Assuntos

RESUMO

Objective: the development of new drugs against Methicillin-resistant Staphylococcus aureus is a priority to the World Health Organization. So, the objective of this study was to evaluate the antibacterial activity and toxicity of 5-bromo-3-((4-methoxyphenyl) sulfenyl)-1H-indole (3b) against MRSA. Methods: minimum inhibitory concentration (MIC) of 3b was determined against S. aureus ATCC 29213 and 43 clinical isolates. The time-kill assay was performed for 9 isolates. Analysis of variance followed by the post hoc Bonferroni test was used for the statistical tests. Results and conclusions: the MIC50 and MIC90 of 3b were 4 µg.mL-1 and 16 µg.mL-1 respectively. In time-kill assay, the 3b showed bactericidal activity to all evaluated isolates at concentrations of 1xMIC and 2xMIC and the re-growth effect was not observed. About the toxicity tests, 3b has not presented cytotoxicity, mutagenicity, or allergenicity. 3b had particularly good activity against MRSA demonstrating high potential for the development of new antimicrobials products.

Objetivo: o desenvolvimento de novos antimicrobianos contra Staphylococcus aureus resistentes à meticilina (MRSA) é uma prioridade para a Organização Mundial da Saúde. Então, o objetivo desse estudo foi avaliar a atividade antibacteriana e a toxicidade do 5-bromo-3-((4-metoxifenil) sulfenil)-1H-indol (3b) contra MRSA. Métodos: a concentração inibitória minima de 3b foi determinada contra S. aureus ATCC 29213 e 43 isolados clínicos. O ensaio de curva de morte foi realizado para nove isolados. Análise de variância seguida pelo teste post hoc Bonferroni foi usada para testes estatísticos. Resultados e conclusões: a MIC50 e MIC90 do 3b foi 4 µg.mL-1 e 16 µg.mL-1, respectivamente. No ensaio de curva de morte, o 3b demonstrou atividade bactericida contra todos os isolados avaliados na concentração de 1xMIC e 2xMIC e o recrescimento não foi observado. Em relação aos testes de toxicidade, 3b não apresentou citotoxicidade, mutagenicidade ou alergenicidade. 3b apresentou atividade particularmente interessante contra MRSA, demonstrando alto potencial para o desenvolvimento de novos produtos antimicrobianos.

Assuntos

RESUMO

Determination of polymyxins susceptibility by clinical laboratories is a nightmare, mainly because of physicochemical properties of the drug. Elution tests have already been proposed for colistin, but not for polymyxin B. We aimed to evaluate accuracy of Polymyxin B broth disk elution (PBDE) to determine the susceptibility to this drug. We evaluated 196 Enterobacterales (45.9% polymyxin B-resistant). PBDE was done in 15-mL cation-adjusted Mueller-Hinton broth where one polymyxin B disk (300 U) was eluted (2 µg/mL). BMD was performed as reference method. Categorical Agreement (CA), Major Error (ME) and Very Major Error (VME) were 99.5%, 0% and 1.11% (one false-negative K. pneumoniae MIC 4 µg/mL), respectively. As some institutions preferably use polymyxin B over colistin and in some countries colistin are not commercially available, to specifically evaluate polymyxin B is important. PBDE proved to be a cheap and easy to perform methodology to evaluate susceptibility to polymyxin B among Enterobacterales.

Assuntos