In vitro evaluation of synergism of aminoglycosides in combination with carbapenem, ceftazidime/avibactam and polymyxin drugs in multidrug-resistant K. pneumoniae, A. baumannii, and S. marcescens isolates

Introdução: Nos últimos anos e devido à pandemia de COVID-19, um número crescente de isolados de bactérias gram-negativas tem apresentado resistência aos antibióticos. O último relatório da OMS destaca esses microrganismos dentro da categoria crítica, o que representa um desafio importante no tratamento de infecções relacionadas à assistência à saúde, pela escassez de novas drogas terapêuticas. Estudos recentes demonstram que os tratamentos contra organismos multirresistentes possam ser mais eficazes em terapia combinada de antibióticos do que em monoterapia. Objetivo: Avaliar o sinergismo in vitro dos aminoglicosídeos com outros antimicrobianos em bactérias gram-negativas multirresistentes de linhagens distintas e com mecanismos de resistência variados. Materiais e Métodos: Foram estudados 72 gram-negativos multirresistentes: 40 Klebsiella pneumoniae, 20 Acinetobacter baumannii e 12 Serratia marcescens. Os microrganismos foram isolados de sangue, urina, tecido nervoso e aspirado traqueal e foram identificados no sistema automatizado Vitek-2, por PCR e por sequenciamento de genoma completo. Os métodos de disco aproximação e epsilométrico (E-test) foram utilizados para avaliar o sinergismo in vitro entre os aminoglicosídeos (amicacina e gentamicina) com colistina, meropenem e ceftazidima/avibactam. Resultados: Em 72,5% (29/40) dos isolados de K. pneumoniae se evidenciou efeito sinérgico na combinação entre amicacina com colistina. Nos isolados de A. baumannii foi observado maior efeito sinérgico em 55% (11/20) na combinação entre amicacina com ceftazidima/avibactam. Nos isolados de S. marcescens foi observado efeito sinérgico em 75,0% (9/12) na combinação de amicacina com meropenem. Entretanto, em 17% (2/12) das combinações de amicacina com colistina e 25% (3/12) entre gentamicina com colistina se evidenciou efeito de antagonismo. Houve concordâncias muito boa e boa nas combinações avaliadas de amicacina com colistina e com ceftazidima/avibactam, e amicacina com meropenem, respectivamente. Nos testes de associação entre o perfil de sensibilidade e o efeito de sinergismo se obteve associação estatisticamente significativa (p < 0,05), de acordo com cada combinação avaliada. Os isolados de K. pneumoniae e A. baumannii foram frequentemente carreadores de genes de resistência aos carbapenêmicos, como blaKPC-2 (86%), blaOXA-23 (80%), respectivamente, e outros mecanismos, como enzimas modificadoras de aminoglicosídeos (EMAs) (98%). A avaliação entre os genes de resistência aos aminoglicosídeos e -lactâmicos mostraram associação com níveis altos da concentração inibitória mínima (CIM) nos aminoglicosídeos. Além disso, a presença de algumas EMAs combinados com -lactamases foram uma causa significativa da perda de sinergismo na atividade in vitro. Conclusão: Em nosso estudo, os aminoglicosídeos demostraram maior efeito sinérgico combinado com colistina e ceftazidima/avibactam, o que suscita a possibilidade de ser um esquema a ser estudado clinicamente como uma alternativa à monoterapia em infecções por bactérias multirresistentes. Os métodos de sinergismo avaliados utilizando E-test e disco aproximação apresentaram ótimo desempenho e concordância, tornando-se os métodos mais eficazes e úteis na hora de analisar associação entre os antibióticos no laboratório de microbiologia convencionalIntroduction: In recent years, and due to the COVID-19 pandemic, an increasing number of Gram-negative bacterial isolates have been found to exhibit antibiotic resistance. The latest report from the World Health Organization (WHO) highlights these microorganisms as part of the critical category, which poses a significant challenge in the treatment of healthcare-associated infections, given the scarcity of new therapeutic drugs. Recent studies demonstrate that treatments against multidrug-resistant organisms may be more effective through combination antibiotic therapy rather than monotherapy. Objective: To evaluate the in vitro synergistic effects of aminoglycosides in combination with other antimicrobials against multidrug-resistant gram-negative bacteria from diverse lineages and with various resistance mechanisms. Materials and Methods: A total of 72 multidrug-resistant Gram-negative bacteria were included: 40 Klebsiella pneumoniae, 20 Acinetobacter baumannii, and 12 Serratia marcescens. The microorganisms were isolated from blood, urine, nervous tissue, and tracheal aspirates and were identified using the automated Vitek-2 system, PCR, and whole-genome sequencing. Disk approximation and E-test methods were employed to assess the in vitro synergy between aminoglycosides (amikacin and gentamicin) and colistin, meropenem, and ceftazidime/avibactam. Results: A synergistic effect was observed in 72.5% (29/40) of K. pneumoniae isolates when combining amikacin with colistin. In A. baumannii isolates, a higher synergistic effect was observed in 55% (11/20) with the combination of amikacin and ceftazidime/avibactam. S. marcescens isolates exhibited a greater synergistic effect in 75.0% (9/12) with the combination of amikacin and meropenem. However, in 17% (2/12) of the amikacin-colistin combinations and 25% (3/12) of the gentamicin-colistin combinations, an antagonistic effect was observed. There were very good and good agreements in the evaluated combinations of amikacin with colistin and with ceftazidime/avibactam, and amikacin with meropenem, respectively. Significant statistical associations (p < 0,05) were found between the sensitivity profile and the synergistic effect for each combination evaluated. K. pneumoniae and A. baumannii isolates frequently harbored carbapenem resistance genes, such as blaKPC-2 (86%) and blaOXA-23 (80%), respectively, along with other mechanisms like aminoglycoside-modifying enzymes (98%). Evaluation of aminoglycoside and -lactam resistance genes revealed an association with high minimum inhibitory concentration (MIC) levels in aminoglycosides. Furthermore, the presence of certain AMEs combined with -lactamases was a significant cause of synergy loss in in vitro activity. Conclusion: In our study, aminoglycosides demonstrated a greater synergistic effect with colistin and ceftazidime/avibactam, suggesting they could be considered as an alternative to monotherapy for infections caused by multidrug-resistant bacteria. The synergy evaluation methods using E-test and disk approximation showed excellent performance and agreement, making them the most effective and useful methods for analyzing antibiotic associations in conventional microbiology laboratorie

Are mobile phones part of the chain of transmission of SARS-CoV-2 in hospital settings?

Mobile phones (MPs) have become an important work tool around the world including in hospitals. We evaluated whether SARS-CoV-2 can remain on the surface of MPs of first-line healthcare workers (HCW) and also the knowledge of HCWs about SARS-CoV-2 cross-transmission and conceptions on the virus survival on the MPs of HCWs. A cross-sectional study was conducted in the COVID-19 Intensive Care Unit of a teaching hospital. An educational campaign was carried out on cross-transmission of SARS-CoV-2, and its permanence in fomites, in addition to the proper use and disinfection of MPs. Herewith an electronic questionnaire was applied including queried conceptions about hand hygiene and care with MP before and after the pandemic. The MPs were swabbed with a nylon FLOQ Swab™, in an attempt to increase the recovery of SARS-CoV-2. All MP swab samples were subjected to SARS-CoV-2 RT-PCR; RT-PCR positive samples were subjected to viral culture in Vero cells (ATCC® CCL-81™). Fifty-one MPs were swabbed and a questionnaire on hand hygiene and the use and disinfection of MP was applied after an educational campaign. Most HCWs increased adherence to hand hygiene and MP disinfection during the pandemic. Fifty-one MP swabs were collected and two were positive by RT-PCR (4%), with Cycle threshold (Ct ) values of 34-36, however, the cultures of these samples were negative. Although most HCWs believed in the importance of cross-transmission and increased adherence to hand hygiene and disinfection of MP during the pandemic, SARS-CoV-2 RNA was detected in MPs. Our results suggest the need for a universal policy in infection control guidelines on how to care for electronic devices in hospital settings

PERFIL DE PROTEÍNA MALDI-TOF MS DE AMOSTRAS DE URINA COMO FATOR PREDITIVO DE GRAVIDADE DA COVID-19 USANDO MACHINE LEARNING

Introdução/Objetivos: O prognóstico da COVID-19 é uma etapa essencial para aumentar a sobrevida do paciente e desempenha um papel importante na alocação de recursos de saúde. A detecção precoce da COVID-19 grave requer técnicas não invasivas, rápidas, de baixo custo e precisas. A proteômica já é descrita na literatura como capaz de detectar padrões para COVID-19 grave, entretanto o uso de amostras pouco invasivas como urina foram pouco exploradas. Neste trabalho utilizamos a proteômica MALDI-TOF MS de amostra de urina combinada com dados clínicos e aprendizado de máquina para predizer gravidade da COVID-19. Métodos: Coorte prospectiva de 372 pacientes hospitalizados com COVID-19 confirmado, realizada no Hospital das Clínicas da FMUSP e no hospital Sírio Libanês, durante o período de julho de 2020 e setembro de 2021. 365 pacientes com até 15 dias de sintomas respiratórios foram incluídos. Amostras de urina foram coletadas, centrifugadas e o sobrenadante estocado a -80°C até o momento de análise. Para obtenção do proteoma por MALDI-TOF MS um total de 500µL de urina foram filtrados (filtro Amicon de 10 kD), dessalinizados (utilizando coluna C18) e submetidos a MALDI-TOF MS, usando uma matriz HCCA. Os arquivos brutos foram pré-processados no R, submetidos às etapas de transformação de dados, normalização, suavização e identificação de picos. A normalidade dos picos identificados foi testada e um teste Wilcoxon rank-sum foi realizado para filtrar os picos proteicos mais relevantes. Os picos resultantes foram usados para treinar um modelo de aprendizado de máquina para classificação de amostras entre condições leves e graves com e sem dados clínicos. Como critério de gravidade, foram considerados necessidade de ventilação mecânica, internação, óbito e marcadores de função renal como ureia e creatinina. Resultados: O modelo de floresta aleatória treinado apenas com o MALDI-TOF MS alcançou um AUC-ROC de 0,760, com precisão, sensibilidade e especificidade de 0,73, 0,77 e 0,69, respectivamente na predição de gravidade da COVID-19. A adição de dados clínicos aos dados proteômicos resultou em um AUC-ROC de 0,827 e sensibilidade e especificidade de 0,81 e 0,87, respectivamente. Conclusões: O perfil proteico por MALDI-TOF MS demonstrou ter potencial para prognóstico de COVID-19; no entanto, a alta variabilidade do proteoma da urina prejudicou o desempenho do modelo. A adição de dados clínicos demonstrou aumentar o desempenho do modelo na classificação da amostra

Virulomic Analysis of Multidrug-Resistant Klebsiella pneumoniae Isolates and Experimental Virulence Model Using Danio rerio (Zebrafish)

This study evaluates a possible correlation between multidrug-resistant Klebsiella pneumoniae strains and virulence markers in a Danio rerio (zebrafish) model. Whole-genome sequencing (WGS) was performed on 46 strains from three Brazilian hospitals. All of the isolates were colistin-resistant and harbored blaKPC-2. Ten different sequence types (STs) were found; 63% belonged to CC258, 22% to ST340, and 11% to ST16. The virulence factors most frequently found were type 3 fimbriae, siderophores, capsule regulators, and RND efflux-pumps. Six strains were selected for a time-kill experiment in zebrafish embryos: infection by ST16 was associated with a significantly higher mortality rate when compared to non-ST16 strains (52% vs. 29%, p = 0.002). Among the STs, the distribution of virulence factors did not differ significantly except for ST23, which harbored a greater variety of factors than other STs but was not related to a higher mortality rate in zebrafish. Although several virulence factors are described in K. pneumoniae, our study found ST16 to be the only significant predictor of a virulent phenotype in an animal model. Further research is needed to fully understand the correlation between virulence and sequence types

Multicenter Diagnostic Evaluation of OnSite COVID-19 Rapid Test (CTK Biotech) among Symptomatic Individuals in Brazil and the United Kingdom

The COVID-19 pandemic has given rise to numerous commercially available antigen rapid diagnostic tests (Ag-RDTs). To generate and to share accurate and independent data with the global community requires multisite prospective diagnostic evaluations of Ag-RDTs. This report describes the clinical evaluation of the OnSite COVID-19 rapid test (CTK Biotech, CA, USA) in Brazil and the United Kingdom. A total of 496 paired nasopharyngeal (NP) swabs were collected from symptomatic health care workers at Hospital das Clínicas in São Paulo, Brazil, and 211 NP swabs were collected from symptomatic participants at a COVID-19 drive-through testing site in Liverpool, United Kingdom. Swabs were analyzed by Ag-RDT, and results were compared to quantitative reverse transcriptase PCR (RT-qPCR). The clinical sensitivity of the OnSite COVID-19 rapid test in Brazil was 90.3% (95% confidence interval [CI], 75.1 to 96.7%) and in the United Kingdom was 75.3% (95% CI, 64.6 to 83.6%). The clinical specificity in Brazil was 99.4% (95% CI, 98.1 to 99.8%) and in the United Kingdom was 95.5% (95% CI, 90.6 to 97.9%). Concurrently, analytical evaluation of the Ag-RDT was assessed using direct culture supernatant of SARS-CoV-2 strains from wild-type (WT), Alpha, Delta, Gamma, and Omicron lineages. This study provides comparative performance of an Ag-RDT across two different settings, geographical areas, and populations. Overall, the OnSite Ag-RDT demonstrated a lower clinical sensitivity than claimed by the manufacturer. The sensitivity and specificity from the Brazil study fulfilled the performance criteria determined by the World Health Organization, but the performance obtained from the UK study failed to do. Further evaluation of Ag-RDTs should include harmonized protocols between laboratories to facilitate comparison between settings. IMPORTANCE Evaluating rapid diagnostic tests in diverse populations is essential to improving diagnostic responses as it gives an indication of the accuracy in real-world scenarios. In the case of rapid diagnostic testing within this pandemic, lateral flow tests that meet the minimum requirements for sensitivity and specificity can play a key role in increasing testing capacity, allowing timely clinical management of those infected, and protecting health care systems. This is particularly valuable in settings where access to the test gold standard is often restricted