Assessment of three antibiotic combination regimens against Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

Genotyping of paired KPC-producing Klebsiella pneumoniae isolates with and without divergent polymyxin B susceptibility profiles.

Polymyxins are still used mainly in treating infections caused by carbapenem-resistant Klebsiella pneumoniae worldwide. The most frequent mechanism of acquired resistance to polymyxins in Gram-negative bacilli is the occurrence of mutations in chromosomal genes regulating operons responsible for lipopolysaccharide modification. As we observed at Santa Casa de São Paulo hospital the occurrence of infections caused by isolates resistant to polymyxins in patients previously treated with this antimicrobial, and new infections caused by the same polymyxin-susceptible species, in this study, we aimed to determine the clonality of consecutive K. pneumoniae isolates from the same patients and characterize the molecular determinants of polymyxin resistance in paired or clonal isolates. A total of 24 pairs and one trio of K. pneumoniae isolates were included in this study. Species identification was achieved by mass spectrometry and multiplex PCR. Polymyxin B minimal inhibitory concentrations were determined by broth microdilution. Clonality was evaluated using pulsed-field gel electrophoresis. The presence of insertions in mgrB gene was tested by PCR, and mutations on pmrA, pmrB, phoP, and phoQ were evaluated by PCR and complete nucleotide sequencing. A fraction of 23.8% of strains resistant to polymyxin B had an insertion in mgrB. Amino acid substitution F204L in PmrB may be implicated in polymyxin resistance. Substitutions T246A and R256G in PmrB were not implicated in polymyxin resistance. In this study, polymyxin resistance after a first susceptible isolate was detected was most frequently due to an infection caused by a distinct clone.

The impact of sonication cultures when the diagnosis of prosthetic joint infection is inconclusive.

BACKGROUND: In the absence of a gold standard criterion for diagnosing prosthetic joint infections (PJI), sonication of the removed implant may provide superior microbiological identification to synovial fluid and peri-implant tissue cultures. The aim of this retrospective study was to assess the role of sonication culture compared to tissue cultures for diagnosing PJI, using different consensus and international guidelines for PJI definition. METHODS: Data of 146 patients undergoing removal of hip or knee arthroplasties between 2010 and 2018 were retrospectively reviewed. The International Consensus Meeting (ICM-2018), Musculoskeletal Infection Society (MSIS), Infectious Diseases Society of America (IDSA), the European Bone and Joint Infection Society (EBJIS), and a modified clinical criterion, were used to compare the performance of microbiological tests. McNemar´s test and proportion comparison were employed to calculate p-value. RESULTS: Overall, 56% (82/146) were diagnosed with PJI using the clinical criteria. Out of these cases, 57% (47/82) tested positive on tissue culture and 93% (76/82) on sonication culture. Applying this clinical criterion, the sensitivity of sonication fluid and tissue cultures was 92.7% (95% CI: 87.1%- 98.3%) and 57.3% (95% CI: 46.6%-68.0%) (p<0.001), respectively. When both methods were combined for diagnosis (sonication and tissue cultures) sensitivity reached 96.3% (95% CI: 91.5%-100%). Sonication culture and the combination of sonication with tissue cultures, showed higher sensitivity rates than tissue cultures alone for all diagnostic criteria (ICM-18, MSIS, IDSA and EBJIS) applied. Conversely, tissue culture provided greater specificity than sonication culture for all the criteria assessed, except for the EBJIS criteria, in which sonication and tissue cultures specificity was 100% and 95.3% (95% CI: 87.8-100%), respectively (p = 0.024). CONCLUSIONS: In a context where diagnostic criteria available have shortcomings and tissue cultures remain the gold standard, sonication cultures can aid PJI diagnosis, especially when diagnostic criteria are inconclusive due to some important missing data (joint puncture, histology).

Microbiological diagnosis of intramedullary nailing infection: comparison of bacterial growth between tissue sampling and sonication fluid cultures.

BACKGROUND: Intramedullary nailing (IMN) has been frequently indicated to treat long bone open and closed fractures, but IMN infection (IMNI) may have devastating consequences. Sonication has been regarded as an important add-on for microbial identification on a variety of orthopaedic implant-associated infections, but its role in the IMNI is poorly studied. We aim at evaluating the accuracy obtained by conventional peri-implant tissue culture (TC) samples with sonication fluid cultures (SCs) of IMNI. METHODS: Longitudinal prospective cohort study ongoing since June 2014, which included patients with indication for IMN removal due to any reason. Clinical diagnosis of INMI was defined according to publication addressing fracture-related infections. Minimal of two samples from TC were cultured. SCs followed the protocol previously published. Statistical analysis was performed using McNemar's test for related proportions. RESULTS: We included 54 patients submitted to IMN retrieval, of whom 47 presenting clinical signs of IMNI. Sensitivity for detecting microorganisms using TC and SC was 89.4% (42/47) and 97.6% (40/41), and specificity was 71.4% (5/7) for both TC and SC (p = 1.00). Positive and negative predictive values for TC and SC were 95.5% (42/44), 95.2% (40/42), 50% (5/10), and 83.3% (5/6), respectively. The most frequent organisms isolated in both TC and SC were Staphylococcus aureus, S. epidermidis, and Enterococcus sp. Polymicrobial infection was diagnosed in 14.8% (8/54) and 25% (12/48) by TC and SC, respectively (p = 0.19). CONCLUSION: Sonication fluid and tissue samples presented similar accuracy on the diagnosis of IMNI, but SC was advantageous of detecting polymicrobial infection.

Panton-Valentine Positive Staphylococcus aureus in Community-Acquired and Hospital-Acquired Pediatric Infections.

From July 2009 to July 2015, Staphylococcus aureus isolated from pediatric sterile sites were selected. Polymerase chain reaction was used to detect mecA and lukS-PV/lukF-PV genes. The rate of methicillin-resistant Staphylococcus aureus was 37.7%. Ten isolates had the lukS-PV/lukF-PV genes, 2 of which were methicillin-resistant Staphylococcus aureus. Skin and soft tissues infections were significantly associated with lukS-PV/lukF-PV positive isolates, P = 0.008.

Hospital-Acquired Infections in Children: A Latin American Tertiary Teaching Hospital 5-Year Experience.

Hospital-acquired infections account for high mortality rates and hospital costs. We analyzed pediatric data from a tertiary teaching hospital and found that most of the cases occurred in the intensive care unit and had significant association with invasive devices. Bloodstream infections were the main site of infection, and Gram-negative bacteria were the predominant etiology.

Evaluation of aflibercept and ziv-aflibercept binding affinity to vascular endothelial growth factor, stability and sterility after compounding.

PURPOSE: To investigate the binding affinity, stability, and sterility of aflibercept and ziv-aflibercept to vascular endothelial growth factor (Holash et al. in Proc Natl Acad Sci USA 99(17):11393-11398, 2002. 10.1073/pnas.172398299) after compounding and storage for up to 28 days at 4 °C and - 8 °C. METHODS: Tuberculin-type 1-mL syringes were prepared containing aflibercept (40 mg/mL) and ziv-aflibercept (25 mg/mL). Samples were stored at 4 °C and - 8 °C for 0, 14, and 28 days and evaluated for the binding affinity of anti-VEGF to VEGF and stability using enzyme-linked immunosorbent assays. The evaluation of sample sterility was performed. RESULTS: Laboratory trials with aflibercept and ziv-aflibercept showed preservation of the drug-binding capability to recombinant VEGF when stored in plastic syringes for up to 28 days at 4 °C and - 8 °C. No significant decrease in mass or concentration were observed. Microbiologic evaluations did not detect contamination in the syringes. CONCLUSIONS: The current study corroborates that compounded anti-VEGF drugs aflibercept and ziv-aflibercept do not loose stability or binding affinity and do not become contaminated if prepared under sterile conditions and stored at 4 °C or - 8 °C for 14 or 28 days.

Avaliação da segurança do processamento de fresas intramedulares flexíveis para cirurgia ortopédica / Safety assessment of reprocessing of flexible intramedullary bone reamers for orthopedic surgery / Evaluación de la seguridad del procesamiento de fresas intramedulares flexibles para cirugía ortopédica

Objetivos: Avaliar a eficácia de um procedimento operacional padrão para limpeza de fresas intramedulares flexíveis, bem como o alcance da esterilidade, e evi-denciar a citotoxicidade da sujidade residual de uma fresa flexível utilizada na prática assistencial. Métodos: Fresas intramedulares flexíveis foram pesadas antes do processamento, após contaminação desafio e depois da limpeza. Elas foram contaminadas com Soil Test™, suspensão de Geobacillus stearothermophilus, na concentração de 106 UFC/mL, e farinha de osso bovino. Após processamento, as amostras foram incubadas em meio de cultura por 21 dias. A sujidade residual de uma fresa utilizada na prática foi submetida ao teste de citotoxicidade in vitro. Resultados: As amostras, embora esterilizadas, apontaram acúmulo de sujidade e o processamento foi ineficaz. A sujidade residual apresentou efeito citotóxico. Conclusão: Recomenda-se que o design flexível das fresas seja descontinuado pela insegurança no processamento.

Objectives: To assess the efficacy of a standard operational procedure to clean flexible intramedullary bone reamers, as well as the sterilization level, and to show the cytotoxicity of the residual dirtiness of a flexible reamer used in care practice. Methods: Flexible intramedullary bone reamers were weighed before processing, after challenge contamination and after cleaning. They were contaminated with the Soil Test™, Geobacillus stearothermo-philus suspension, in the concentration of 106 cfu/ml, and bovine bone flour. After processing, the samples were inoculated into a culture medium and incubated for 21 days. Residual dirtiness of a flexible intramedullary bone reamer used in practice was submitted to in vitro cytotoxicity test. Results: Despite being sterilized, the samples indicate to accumulated dirtiness and the processing was inefficient. Residual dirtiness presented a cytotoxic effect. Conclusion: It is recommended that the flexible design of reamers is discontinued by the lack of safety of reprocessing.

Objetivos: Evaluar la eficacia de un procedimiento operacional estándar para limpieza de fresas intramedulares flexibles, así como el alcance de la esterilidad, y evidenciar la citotoxicidad de la suciedad residual de una fresa flexible utilizada en la práctica asistencial. Métodos: Fresas intramedulares flexibles fueron pesadas antes del procesamiento, tras contaminación desafío y después de la limpieza. Fueron contaminadas con Soil Test™, suspensión de Geobacillus stearothermophilus, en la concentración de 106 UFC/mL, y harina de hueso bovino. Tras el procesamiento, las muestras fueron incuba-das en medio de cultura por 21 días. La suciedad residual de una fresa utilizada en la práctica fue sometida al test de citotoxicidad in vitro. Resultados: Las muestras, aunque esterilizadas, apuntaron acumulación de suciedad y el procesamiento fue ineficaz. La suciedad residual presentó efecto citotóxico. Conclusión: Se recomienda que el design flexible de las fresas sea descontinuado por la inseguridad en el procesamiento.

Evaluation of the effectiveness of manual and automated dialyzers reprocessing after multiple reuses.

A cross-sectional study was conducted to evaluate the effectiveness of manual and automated dialyzer reprocessing. Dialyzers were filled with fluid thioglycollate medium from blood and dialysate chambers after being reprocessed and chemically sterilized with 0.2% peracetic acid. They were incubated for 14 days at 35°C ± 2°C, and microbiologic analysis was performed. Microorganisms were identified in 3 of the 11 samples (27.3%) from the blood chambers: Sphingomonas paucimobilis (2/3) and Penicillium spp (1/3) and in 11 of the 11 samples (100%) from the dialysate chambers: S paucimobilis (7/11), Stenotrophomonas maltophilia (4/11), Pseudomonas aeruginosa (3/11), Candida spp (1/11), and Acinetobacter baumannii (1/11). Of the 4 manually reprocessed dialyzers, gram-positive bacillus were identified in 1 sample (25%) from the blood chamber, and Bacillus spp and Burkholderia spp were identified in 1 sample (25%) from the dialysate chamber. The dialyzers reprocessing can pose risks safety because of exposure patient to microorganisms.

Concentration of Sonication Fluid through Centrifugation Is Superior to Membrane Filtration for Microbial Diagnosis of Orthopedic Implant-Associated Infection.

Microbial identification of orthopedic implant-associated infections using sonication fluid (SF) submitted to a concentration step by membrane filtration (SMF) was compared with the standard centrifugation (SC) method. Among 33 retrieved infected implants, sonication identified microorganisms in 26 (78.8%). The sensitivity of SC was higher than that of SMF (78.8% versus 30.3%; P < 0.001).

Microbial diagnosis of infection and colonization of cardiac implantable electronic devices by use of sonication.

OBJECTIVES: The clinical utility of sonication as an adjunctive diagnostic tool for the microbial diagnosis of cardiac implantable device-associated infections (CIDAIs) was investigated. METHODS: The implants of 83 subjects were investigated, 15 with a CIDAI and 68 without a clinical infection. Clinical data were analyzed prospectively and sonication fluid cultures (83 patients, 100%) and traditional cultures (31 patients, 37.4%) were performed RESULTS: Generator pocket infection and device-related endocarditis were found in 13 (86.7%) and four (26.7%) subjects, respectively. The mean numbers of previous technical complications and infections were higher in the infected patients compared to the non-infected patients (8 vs. 1, p<0.001; 2 vs. 0, p<0.031, respectively). The sensitivity and specificity for detecting CIDAI was 73.3% (11/15) and 48.5% (33/68) for sonication fluid culture, and 26.7% (4/15) and 100% (16/16) for traditional culture (p<0.001), respectively. A higher number of organisms were identified by sonication fluid than by tissue culture (58 vs. 4 specimens; p<0.001). The most frequent organisms cultured were Gram-positive cocci (66.1%), mainly coagulase-negative staphylococci (35.5%). Thirty-five (51.5%) non-infected subjects were considered colonized due to the positive identification of organisms exclusively through sonication fluid culture. CONCLUSIONS: Sonication fluid culture from the removed cardiac implants has the potential to improve the microbiological diagnosis of CIDAIs.

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.

Improved diagnosis of infection associated with osteosynthesis by use of sonication of fracture fixation implants.

Previous studies have shown that sonication fluid cultures from removed orthopedic devices improved the microbiological diagnosis of orthopedic implant-associated infections; however, few of these investigations have applied sonication to the removed fracture fixation devices to evaluate its utility for the diagnosis of osteosynthesis-associated infection (OAI). We compared sonication fluid to conventional tissue cultures from 180 subjects with different sizes of plates and screws (n = 156), spinal implants (n = 26), and intramedullary nails (n = 3), of whom 125 and 55 subjects had OAI and noninfected osteosynthesis (NIO), respectively. The sensitivity for detecting OAI was 90.4% for sonication fluid culture and 56.8% for periprosthetic tissue cultures (P < 0.05), and the specificities were 90.9% and 96.4%, respectively. Sonication fluid culture detected more pathogens than peri-implant tissue culture (113 versus 71; P < 0.001), while polymicrobial infections were diagnosed by sonication fluid cultures and tissue cultures in 20.8% and 8% (P < 0.001), respectively. Microbiological diagnosis was achieved exclusively by sonication fluid cultures for 47 (90.4%) subjects, and among them, 18 (38.3%) had previously received antibiotics, whereas in five (9.6%) infected subjects, tissue culture was positive and the sonication fluid culture was negative. Among 39 (31.2%) OAI cases receiving antibiotics, the identification of the organisms occurred in 38.5% and 82.1% of the tissue and sonication fluid cultures, respectively (P < 0.049). We demonstrated that sonication fluid culture from removed osteosyntheses has the potential for improving the microbiological diagnosis of OAI.

Evaluation of the in vitro antimicrobial activity of an ethanol extract of Brazilian classified propolis on strains of Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is one of the most frequent causes of hospital acquired infections. With the increase in multiple drug resistant strains, natural products such as propolis are a stratagem for new product discovery. The aims of this study were: to determine the in vitro antimicrobial activity of an ethanol extract of propolis; to define the MIC50 and MIC90 (Minimal Inhibitory Concentration - MIC) against 210 strains of S. aureus; to characterize a crude sample of propolis and the respective ethanol extract as to the presence of predetermined chemical markers. The agar dilution method was used to define the MIC and the high performance liquid chromatography (HPLC) method was used to characterize the samples of propolis. MIC results ranged from 710 to 2,850 µg/mL. The MIC50 and MIC90 for the 210 strains as well as the individual analysis of American Type Culture Collection (ATCC) strains of Methicillin-susceptible Staphylococcus aureus (MSSA) and Methicillin-resistant Staphylococcus aureus (MRSA) were both 1,420 µg/mL. Based on the chromatographic analysis of the crude sample and ethanol extracted propolis, it was concluded that propolis was a mixture of the BRP (SP/MG) and BRP (PR) types. The results obtained confirm an antimicrobial activity in relation to the strains of the S. aureus tested.

Analysis of the microbial load in instruments used in orthopedic surgeries.

BACKGROUND: Because of advances in technology, the number of orthopedic surgeries, mainly hip and knee replacement surgeries, has increased, with a total of 150,000 prosthetic surgeries estimated per year in the United States and 400,000 worldwide. METHODS: We used an exploratory cross-sectional study, with a quantitative approach to determine the microbial load in instruments used in orthopedic surgeries, quantifying and identifying the microbial growth genus and species, according to the surgical potential of contamination that characterizes the challenge faced by the Material and Sterilization Center at the Institute of Orthopedics and Traumatology of Hospital das Clinicas of the School of Medicine of the University of Sao Paulo, Brazil.The orthopedic surgical instruments were immersed, after their use, in sterilized distilled water, sonicated in an ultrasonic washer, and posteriorly agitated. Subsequently, the wash was filtrated through a 0.45-mum membrane and incubated in aerobic and anaerobic mediums and in medium for fungi and yeasts. RESULTS: In clean surgeries, 47% of the instruments were contaminated; in contaminated surgeries, 70%; and, in infected surgeries, 80%. Regardless of the contamination potential of the surgeries, the highest quantitative incidence of microorganism recovery was located in the 1 to 100 colony-forming unit range, and 13 samples presented a microbial growth potential >300 colony-forming units. Regardless of the contamination potential of the surgeries, there was a convergence in the incidence of negative-coagulase Staphylococcus growth (28%, clean surgeries; 32%, contaminated surgeries; and 29%, infected surgeries) and Staphylococcus aureus (28%, contaminated surgeries; and 43%, infected surgeries). CONCLUSION: Most of the microorganisms recovered from the analyzed instruments (78%) were vegetative bacteria that presented their death curve at around 80 degrees C, characterizing a low challenge considering the processes of cleaning and sterilization currently employed by the Material and Sterilization Center. Fewer microorganisms were recovered from instruments used in clean surgeries in comparison with those used in contaminated and infected surgeries.