Nanosphere's Verigene(®) Blood Culture Assay to Detect Multidrug-Resistant Gram-Negative Bacterial Outbreak: A Prospective Study on 79 Hematological Patients in a Country with High Prevalence of Antimicrobial Resistance. - Related Documents




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223501.0000Nanosphere's Verigene(®) Blood Culture Assay to Detect Multidrug-Resistant Gram-Negative Bacterial Outbreak: A Prospective Study on 79 Hematological Patients in a Country with High Prevalence of Antimicrobial Resistance. Infections are a major cause of morbidity and mortality in hematological patients. We prospectively tested a new molecular assay (Verigene(®)) in 79 consecutive hematological patients, with sepsis by gram-negative bacteria. A total of 82 gram-negative microorganisms were isolated by blood cultures, of which 76 cases were mono-microbial. Considering the bacteria detectable by the system, the concordance with standard blood cultures was 100%. Resistance genes were detected in 20 of the isolates and 100% were concordant with the phenotypic antibiotic resistance. Overall, this new assay correctly identified 66/82 of all the gram-negative pathogens, yielding a general sensitivity of 80.5%, and providing information on genetic antibiotic resistance in a few hours. This new molecular assay could ameliorate patient management, resulting in a more rational use of antibiotics.201934595420
223610.9998Development of a Multiplex PCR Platform for the Rapid Detection of Bacteria, Antibiotic Resistance, and Candida in Human Blood Samples. The diagnosis of bloodstream infections (BSIs) still relies on blood culture (BC), but low turnaround times may hinder the early initiation of an appropriate antimicrobial therapy, thus increasing the risk of infection-related death. We describe a direct and rapid multiplex PCR-based assay capable of detecting and identifying 16 bacterial and four Candida species, as well as three antibiotic-resistance determinants, in uncultured samples. Using whole-blood samples spiked with microorganisms at low densities, we found that the MicrobScan assay had a mean limit of detection of 15.1 ± 3.3 CFU of bacteria/Candida per ml of blood. When applied to positive BC samples, the assay allowed the sensitive and specific detection of BSI pathogens, including bla(KPC)-, mecA-, or vanA/vanB-positive bacteria. We evaluated the assay using prospectively collected blood samples from patients with suspected BSI. The sensitivity and specificity were 86.4 and 97.0%, respectively, among patients with positive BCs for the microorganisms targeted by the assay or patients fulfilling the criteria for infection. The mean times to positive or negative assay results were 5.3 ± 0.2 and 5.1 ± 0.1 h, respectively. Fifteen of 20 patients with MicrobScan assay-positive/BC-negative samples were receiving antimicrobial therapy. In conclusion, the MicrobScan assay is well suited to complement current diagnostic methods for BSIs.201931799215
223020.9998Rapid detection of gram-negative antimicrobial resistance determinants directly from positive blood culture broths using a multiplex PCR system. Currently available rapid blood culture diagnostics detect few gram-negative resistance determinants, limiting their clinical utility. We prospectively evaluated the prototype BIOFIRE FILMARRAY Antimicrobial Resistance (AMR) Panel, a rapid multiplex PCR test that detects 31 AMR genes, on residual positive blood culture broths from patients with gram-negative bacteremia due to five target organisms at a New York City hospital. Predicted antimicrobial resistance based on the AMR Panel was compared to results from broth microdilution testing of bloodstream isolates recovered in culture. A simulated stewardship study assessed opportunities for the optimization of therapy if the AMR Panel results had been available for patient care in real time. We enrolled 148 patients with gram-negative bacteremia (Escherichia coli, n = 75; Klebsiella pneumoniae, n = 44; Pseudomonas aeruginosa, n = 17; Enterobacter cloacae complex, n = 9; and Acinetobacter baumannii, n = 3). The sensitivity of the AMR Panel for predicting antimicrobial resistance was ≥90% for 10/14 antimicrobial agents in E. coli and for 10/16 agents in K. pneumoniae. Specificity was ≥90% for 15/17 agents in E. coli and for all 16 agents in K. pneumoniae. Performance for other organisms was poor. For E. coli or K. pneumoniae bacteremia, use of the AMR Panel could have led to earlier escalation or de-escalation of β-lactam therapy in a majority of patients compared to what actually occurred. This study demonstrates that a rapid multiplex PCR test with a large menu of AMR genes can be applied to positive blood culture broths to rapidly predict resistance to frontline antimicrobial agents in patients with E. coli or K. pneumoniae bacteremia.IMPORTANCEPatients with gram-negative bacteremia require urgent treatment with antimicrobial agents that are effective against their infecting pathogen. However, conventional laboratory work-up of blood cultures takes days to yield results, and during this time, patients may receive ineffective therapies. We evaluated the prototype BIOFIRE FILMARRAY AMR Panel, an assay that detects 31 genes in gram-negative bacteria that confer resistance to β-lactams, fluoroquinolones, and aminoglycosides in approximately 1 hour, directly from positive blood culture broths, and compared these results to antimicrobial susceptibility testing of isolates recovered in culture. We found that the AMR Panel accurately predicted resistance in Escherichia coli and Klebsiella pneumoniae to most antimicrobials. Moreover, if results from this assay had been used for patient care, there would have been opportunities to optimize antimicrobial prescribing more quickly than using conventional methods. These data demonstrate how novel molecular assays could optimize care for patients with E. coli and K. pneumoniae bacteremia.202541117625
579630.9998Antibiotic treatment algorithm development based on a microarray nucleic acid assay for rapid bacterial identification and resistance determination from positive blood cultures. Rapid diagnosis of bloodstream infections remains a challenge for the early targeting of an antibiotic therapy in sepsis patients. In recent studies, the reliability of the Nanosphere Verigene Gram-positive and Gram-negative blood culture (BC-GP and BC-GN) assays for the rapid identification of bacteria and resistance genes directly from positive BCs has been demonstrated. In this work, we have developed a model to define treatment recommendations by combining Verigene test results with knowledge on local antibiotic resistance patterns of bacterial pathogens. The data of 275 positive BCs were analyzed. Two hundred sixty-three isolates (95.6%) were included in the Verigene assay panels, and 257 isolates (93.5%) were correctly identified. The agreement of the detection of resistance genes with subsequent phenotypic susceptibility testing was 100%. The hospital antibiogram was used to develop a treatment algorithm on the basis of Verigene results that may contribute to a faster patient management.201626712265
223440.9998Clinical relevance of molecular identification of microorganisms and detection of antimicrobial resistance genes in bloodstream infections of paediatric cancer patients. BACKGROUND: Bloodstream infections (BSIs) are the major cause of mortality in cancer patients. Molecular techniques are used for rapid diagnosis of BSI, allowing early therapy and improving survival. We aimed to establish whether real-time quantitative polymerase chain reaction (qPCR) could improve early diagnosis and therapy in paediatric cancer patients, and describe the predominant pathogens of BSI and their antimicrobial susceptibility. METHODS: Blood samples were processed by the BACTEC system and microbial identification and susceptibility tests were performed by the Phoenix system. All samples were screened by multiplex 16 s rDNA qPCR. Seventeen species were evaluated using sex-specific TaqMan probes and resistance genes blaSHV, blaTEM, blaCTX, blaKPC, blaIMP, blaSPM, blaVIM, vanA, vanB and mecA were screened by SYBR Green reactions. Therapeutic efficacy was evaluated at the time of positive blood culture and at final phenotypic identification and antimicrobial susceptibility results. RESULTS: We analyzed 69 episodes of BSI from 64 patients. Gram-positive bacteria were identified in 61 % of the samples, Gram-negative bacteria in 32 % and fungi in 7 %. There was 78.2 % of agreement between the phenotypic and molecular methods in final species identification. The mecA gene was detected in 81.4 % of Staphylococcus spp., and 91.6 % were concordant with the phenotypic method. Detection of vanA gene was 100 % concordant. The concordance for Gram-negative susceptibilities was 71.4 % for Enterobacteriaceae and 50 % for Pseudomonas aeruginosa. Therapy was more frequently inadequate in patients who died, and the molecular test was concordant with the phenotypic susceptibility test in 50 %. CONCLUSIONS: qPCR has potential indication for early identification of pathogens and antimicrobial resistance genes from BSI in paediatric cancer patients and may improve antimicrobial therapy.201627585633
223350.9998Assessment of the multiplex PCR-based assay Unyvero pneumonia application for detection of bacterial pathogens and antibiotic resistance genes in children and neonates. BACKGROUND: Pneumonia is a major healthcare problem. Rapid pathogen identification is critical, but often delayed due to the duration of culturing. Early, broad antibacterial therapy might lead to false-negative culture findings and eventually to the development of antibiotic resistances. We aimed to assess the accuracy of the new application Unyvero P50 based on multiplex PCR to detect bacterial pathogens in respiratory specimens from children and neonates. METHODS: In this prospective study, bronchoalveolar lavage fluids, tracheal aspirates, or pleural fluids from neonates and children were analyzed by both traditional culture methods and Unyvero multiplex PCR. RESULTS: We analyzed specimens from 79 patients with a median age of 1.8 (range 0.01-20.1). Overall, Unyvero yielded a sensitivity of 73.1% and a specificity of 97.9% compared to culture methods. Best results were observed for non-fermenting bacteria, for which sensitivity of Unyvero was 90% and specificity 97.3%, while rates were lower for Gram-positive bacteria (46.2 and 93.9%, respectively). For resistance genes, we observed a concordance with antibiogram of 75% for those specimens in which there was a cultural correlate. CONCLUSIONS: Unyvero is a fast and easy-to-use tool that might provide additional information for clinical decision making, especially in neonates and in the setting of nosocomial pneumonia. Sensitivity of the PCR for Gram-positive bacteria and important resistance genes must be improved before this application can be widely recommended.201829086343
230860.9998Trends of Antibiotic Resistance in Multidrug-Resistant Pathogens Isolated from Blood Cultures in a Four-Year Period. BACKGROUND: Multidrug-resistant organisms cause serious infections with significant morbidity and mortality in the worldwide. These organisms have been identified as urgent and serious threats by CDC. The aim of this study was to determine the prevalence and changes of antibiotic resistance of multidrug-resistant pathogens isolated from blood cultures over a four-year period in a tertiary-care hospital. METHODS: Blood cultures were incubated in a blood culture system. Positive signalling blood cultures were subcultured on 5% sheep-blood agar. Identification of isolated bacteria was performed using conventional or automated identification systems. Antibiotic susceptibility tests were performed by disc diffusion and/or gradient test methods, if necessary, by automated systems. The CLSI guidelines were used for interpretation of antibiotic susceptibility testing of bacteria. RESULTS: The most frequently isolated Gram-negative bacteria was Escherichia coli (33.4%) followed by Klebsiella pneumoniae (21.5%). ESBL positivity was 47% for E. coli, 66% for K. pneumoniae. Among E. coli, K. pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii isolates, carbapenem resistance was 4%, 41%, 37%, and 62%, respectively. Carbapenem resistance of K. pneumoniae isolates has increased from 25% to 57% over the years, and the highest rate (57%) occured during the pandemic period. It is noteworthy that the aminoglycoside resistance in E. coli isolates gradually increased from 2017 to 2021. The rate of methicillin-resistant S. aureus (MRSA) was found to be 35.5%. CONCLUSIONS: Increased carbapenem resistance in K. pneumoniae and A. baumannii isolates is noteworthy, but carbapenem resistance in P. aeruginosa decreased. It is of great importance for each hospital to monitor the increase in resistance in clinically important bacteria, especially isolated from invasive samples, in order to take the necessary precautions in a timely manner. Future studies involving clinical data of patients and bacterial resistance genes are warranted.202337307126
231870.9998Distribution of pathogenic bacteria in lower respiratory tract infection in lung cancer patients after chemotherapy and analysis of integron resistance genes in respiratory tract isolates of uninfected patients. BACKGROUND: We studied the distribution of pathogenic bacteria in lower respiratory tract infection in lung cancer patients after chemotherapy and analyzed the integron resistance genes in respiratory tract isolates of uninfected patients. METHODS: Retrospective analysis was used to select sputum samples from 400 lung cancer patients after chemotherapy admitted in Fuyang People's Hospital from July 2017 to July 2019. Culture, isolation and identification of strains were conducted in accordance with the national clinical examination operating procedures. RESULTS: A total of 134 strains were identified. In 120 patients with pulmonary infection, 114 strains were cultured. Twenty strains of klebsiella pneumoniae were cultured in 280 patients without pulmonary infection. Among the 134 strains, the detection rate of gram-negative bacteria was 79.10%. The first four strains were Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Haemophilus influenzae. The gram-positive bacteria detection rate was 4.47%, mainly Staphylococcus aureus and Streptococcus. The fungus detection rate was 16.42%. The drug sensitivity results showed that the resistance rate of gram-negative bacillus to penicillin and cephalosporin was higher, and were more sensitive to carbapenem, piperacillin tazobactam and cefoperazone sulbactam. Gram-positive cocci were resistant to penicillin, macrolide and clindamycin, and sensitive to linezolid, vancomycin and rifampicin. All strains of fungal culture were candida albicans, which were sensitive to common antifungal drugs. Among the 20 strains of klebsiella pneumoniae cultured in sputum specimens of non-infected patients with lung cancer undergoing chemotherapy, 2 strains were integron-positive strains, and all of them were class I integrons. CONCLUSIONS: Lung cancer patients after chemotherapy have a high resistance to commonly used antimicrobial drugs, so it is necessary to detect the resistance of pathogenic microorganisms in clinical practice. The strains carried by patients with lung cancer without pulmonary infection during chemotherapy can isolate type I integrons, suggesting that the spread of drug resistance at gene level should be closely detected.202032944333
220680.9998Retrospective analysis of pediatric sepsis and the burden of antimicrobial resistance in Duhok, Kurdistan Region of Iraq. Introduction: Sepsis is a life-threatening complication in pediatric patients. This study primarily aimed to investigate sepsis-causing bacteria and their antimicrobial resistance profile and check the change in the antimicrobial resistance trend for some selected bacteria. In addition, we evaluated the incidence of sepsis, the related mortality rate, and the effectiveness and outcome of the treatment regimes in sepsis pediatric patients. Methods: A retrospective analysis was conducted on 4-year data (2018-2021) collected from three intensive care units at the Hevi Pediatric Teaching Hospital. Sepsis screening involved clinical detection and confirmation by blood culture. Results: A total of 520 out of 1,098 (47.35%) blood samples showed positive microbial growth. A decrease in sepsis rate was observed during the COVID-19 pandemic. Coagulase-negative Staphylococci (CoNS) and Klebsiella pneumonia were the most commonly isolated bacteria. A notable variation in the antimicrobial resistance trend was observed among sepsis-causing bacteria. The empirical sepsis treatment recommended by the WHO was ineffective, as certain bacteria exhibited 100% resistance to every antibiotic tested. The mortality rate significantly increased from 1.3% in 2018 to 16.5% in 2021. Discussion: The antimicrobial resistance profile of sepsis causing bacteria is of concerns, indicating a potentially serious situation. Thus, to avoid treatment failure, the monitoring of antimicrobial resistance in pediatric patients is essential.202438469402
577790.9998Rapid Detection of Antimicrobial Resistance Genes in Critically Ill Children Using a Custom TaqMan Array Card. Bacteria are identified in only 22% of critically ill children with respiratory infections treated with antimicrobial therapy. Once an organism is isolated, antimicrobial susceptibility results (phenotypic testing) can take another day. A rapid diagnostic test identifying antimicrobial resistance (AMR) genes could help clinicians make earlier, informed antimicrobial decisions. Here we aimed to validate a custom AMR gene TaqMan Array Card (AMR-TAC) for the first time and assess its feasibility as a screening tool in critically ill children. An AMR-TAC was developed using a combination of commercial and bespoke targets capable of detecting 23 AMR genes. This was validated using isolates with known phenotypic resistance. The card was then tested on lower respiratory tract and faecal samples obtained from mechanically ventilated children in a single-centre observational study of respiratory infection. There were 82 children with samples available, with a median age of 1.2 years. Major comorbidity was present in 29 (35%) children. A bacterial respiratory pathogen was identified in 13/82 (16%) of children, of which 4/13 (31%) had phenotypic AMR. One AMR gene was detected in 49/82 (60%), and multiple AMR genes were detected in 14/82 (17%) children. Most AMR gene detections were not associated with the identification of phenotypic AMR. AMR genes are commonly detected in samples collected from mechanically ventilated children with suspected respiratory infections. AMR-TAC may have a role as an adjunct test in selected children in whom there is a high suspicion of antimicrobial treatment failure.202338136735
2205100.9998Five-year period evaluation of isolated agents and their resistance profiles in intensive care unit patients with malignancy. INTRODUCTION: Patients treated in the intensive care unit (ICU) are usually patients who deteriorated health condition and could have longer hospital stay compared to other patients. Hospital infections are more common in ICU patients. The aim of this study was to evaluate the bacteria and treatment resistance profiles isolated from clinical specimens sent for hospital infections in ICU patients between January 1, 2014 and December 31, 2018. METHODOLOGY: Bacteria isolated from various clinical samples sent for hospital infections in hospitalized patients in the Anesthesia and Reanimation Intensive Care Unit were retrospectively analyzed. RESULTS: Culture positivity was detected in 547 of the sent clinical samples. Eighty Gram-positive bacteria, 389 Gram-negative bacteria and 78 fungi infection were identified in a total of 547 positive cultures. In Gram-positive bacteria, 4 MRSA, 6 VRE and 30 MRCoNS were identified as resistant strains. In Gram-negative bacteria, Acinetobacter spp. was the most culture positive strain with the number of 223. Carbapenem resistance was found in 258 of the Gram-negative bacteria and ESBL positivity was found in 44 of the Gram-negative bacteria strains. CONCLUSIONS: Gram-negative bacteria were the most frequently isolated strain in samples. Recently, colistin resistance has been increasing in Acinetobacter spp. and the increase in carbapenemase enzyme in Escherichia coli, Pseudomonas and Klebsiella species has increased resistance to carbapenems. Knowing the microorganisms that grow in ICUs and their antibiotic resistance patterns may help to prevent contamination of resistant microorganisms by both appropriate empirical antibiotic treatment and more isolation as well as general hygiene standard precautions.202032903237
2227110.9998Prophylactic application of antibiotics selects extended-spectrum β-lactamase and carbapenemases producing Gram-negative bacteria in the oral cavity. Prophylactic administration of broad-spectrum antibiotics in surgery can change the oral microbiome and induce colonization of oral cavity with Gram-negative bacteria including multidrug (MDR) or extensively drug resistant (XDR) organisms which can lead to lower respiratory tract infections. The aim of the study was to analyse the Gram-negative isolates obtained from oral cavity of the mechanically ventilated patients in ICUs, after prophylactic application of antibiotics and their resistance mechanisms and to compare them with the isolates obtained from tracheal aspirates from the same patients. The antibiotic susceptibility was determined by broth dilution method. PCR was applied to detect genes encoding β-lactamases. Marked diversity of Gram-negative bacteria and resistance mechanisms was found. High resistance rates and high rate of bla(CTX-M) and carbapenemase encoding genes (bla(VIM-1) , bla(OXA-48) ) were found among Klebsiella pneumoniae. Pseudomonas aeruginosa was found to harbour bla(VIM) and in one strain bla(PER-1) gene, whereas Acinetobacter baumannii produced OXA-23-like and OXA-24/40-like oxacillinases and was XDR in all except one case. All XDR isolates belong to international clonal lineage II (IC II). The main finding of the study is that the prophlylactic application of antibiotics in surgery intensive care units (ICUs) is associated with the colonization of oral cavity and lower respiratory tract with Gram-negative bacteria. The identity of Gram-negative bacteria in oral cavity reflected those found in endotracheal aspirates leading to conclusion that oral swab as non-invasive specimen can predict the colonization of lower respiratory tract with resistant Gram-negative organisms and the risk for development of ventilator-associated pneumonia.202133896011
5797120.9998PCR-reverse blot hybridization assay for screening and identification of pathogens in sepsis. Rapid and accurate identification of the pathogens involved in bloodstream infections is crucial for the prompt initiation of appropriate therapy, as this can decrease morbidity and mortality rates. A PCR-reverse blot hybridization assay for sepsis, the reverse blot hybridization assay (REBA) Sepsis-ID test, was developed; it uses pan-probes to distinguish Gram-positive and -negative bacteria and fungi. In addition, the assay was designed to identify bacteria and fungi using six genus-specific and 13 species-specific probes; it uses additional probes for antibiotic resistance genes, i.e., the mecA gene of methicillin-resistant Staphylococcus aureus (MRSA) and the vanA and vanB genes of vancomycin-resistant enterococci (VRE). The REBA Sepsis-ID test successfully identified clinical isolates and blood culture samples as containing Gram-positive bacteria, Gram-negative bacteria, or fungi. The results matched those obtained with conventional microbiological methods. For the REBA Sepsis-ID test, of the 115 blood culture samples tested, 47 (40.8%) and 49 (42.6%) samples were identified to the species and genus levels, respectively, and the remaining 19 samples (16.5%), which included five Gram-positive rods, were identified as Gram-positive bacteria, Gram-negative bacteria, or fungi. The antibiotic resistances of the MRSA and VRE strains were identified using both conventional microbiological methods and the REBA Sepsis-ID test. In conclusion, the REBA Sepsis-ID test developed for this study is a fast and reliable test for the identification of Gram-positive bacteria, Gram-negative bacteria, fungi, and antibiotic resistance genes (including mecA for MRSA and the vanA and vanB genes for VRE) in bloodstream infections.201323447637
2213130.9998The distribution and resistance of pathogens causing blood stream infections following liver transplantation: a clinical analysis of 69 patients. BACKGROUND/AIMS: To study the distribution and drug resistance of pathogens causing blood stream infections (BSIs) and provide the evidence for clinical therapy after liver transplantation. METHODOLOGY: Blood samples were processed by the BACTEC 9120 blood culture system. Species identification was performed using the Vitek-2 system. The drug susceptibility of pathogens was performed using the ATB FUNGUS 3 system. RESULTS: One hundred and twenty six episodes of BSIs occurred in 69 patients between January 31, 2003 and January 31, 2014. The gram-positive bacteria emerged as major pathogens and constituted 48.4% of all pathogens (61/126). The most common bacilli were Enterobacter spp and Enterococcus spp followed by S. aureus. The gram-negative bacteria were relatively sensitive to carbapenems and the gram-positive bacteria were relatively sensitive to glycopeptides and oxazolidone antibiotics. The drug resistance of fungi to amphotericin B, flucytosine, voriconazole and caspofungin was not found. CONCLUSION: In liver transplantation, gram-positive bacteria caused BSls more frequently than gram-negative bacteria. The resistance rate of bacteria to antibiotics was high while the rate was low in fungi.201425699372
5779140.9998Development of a One-Step Multiplex qPCR Assay for Detection of Methicillin and Vancomycin Drug Resistance Genes in Antibiotic-Resistant Bacteria. The most common antibiotic-resistant bacteria in Korea are methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Pathogen identification in clinical laboratories can be divided into traditional phenotype- and genotype-based methods, both of which are complementary to each other. The genotype-based method using multiplex real-time polymerase chain reaction (PCR) is a rapid and accurate technique that analyzes material at the genetic level by targeting genes simultaneously. Accordingly, we aimed to develop a rapid method for studying the genetic characteristics of antibiotic-resistant bacteria and to provide an experimental guide for the efficient antibiotic resistance gene analysis of mecA detection for MRSA and vanA or vanB detection for VRE using a one-step multiplex qPCR assay at an early stage of infection. As a result, the sensitivity and specificity of the mecA gene for clinical S. aureus isolates, including MRSA and methicillin-susceptible S. aureus, were 97.44% (95% CI, 86.82-99.87%) and 96.15% (95% CI, 87.02-99.32%), respectively. The receiver operating characteristic area under the curve for the diagnosis of MRSA was 0.9798 (*** p < 0.0001). Therefore, the molecular diagnostic method using this newly developed one-step multiplex qPCR assay can provide accurate and rapid results for the treatment of patients with MRSA and VRE infections.202439452724
2226150.9998Evaluation of the Microbiological Performance and Potential Clinical Impact of New Rapid Molecular Assays for the Diagnosis of Bloodstream Infections. Bloodstream infection (BSI) is a critical medical emergency associated with a high mortality rate. Rapid and accurate identification of the causative pathogen and the results of antimicrobial susceptibility testing are crucial for initiating appropriate antimicrobial therapy. The aim of this study was to evaluate the performance of a new rapid PCR Molecular Mouse System (MMS) for the identification of Gram-negative bacteria (GNB) and GNB resistance genes directly from a positive blood culture (BC). The validation of these rapid multiplex assays was carried out in a real hospital setting. A total of 80 BSI episodes were included in our study and the results were compared with culture-based methods. BC samples in which GNB had previously been detected microscopically and which originated from different hospital wards were analysed. The MMS GNB identification assay achieved a sensitivity of 98.7% and a specificity of 100% for the covered pathogens. In one BC sample, Klebsiella aerogenes was identified at the family level (Enterobacteriaceae) with MMS. However, in three polymicrobial samples, MMS identified bacteria that were not detected by culture-based methods (Klebsiella pneumoniae, K. aerogenes and Stenotrophomonas maltophilia). MMS also showed excellent overall performance in the detection of GNB resistance markers (100% sensitivity and 100% specificity). The type of extended-spectrum beta-lactamase (ESBL) resistance gene identified correctly with MMS was CTX-M-1/9 (n = 17/20), alone or in combination with SHV-type β-lactamase or with the different types of carbapenemase genes. MMS detected one carbapenemase gene of each type (KPC, NDM and OXA-23) and six OXA-48 genes. In addition, the colistin resistance gene mcr-1 was detected in one positive BC with Escherichia coli (E. coli). The time to result was significantly shorter for MMS than for routine culture methods. A retrospective analysis of the patients' medical records revealed that a change in empirical antimicrobial therapy would have been made in around half of the patients following the MMS results. These results support the use of MMS as a valuable complement to conventional culture methods for more rapid BSI diagnosis and adjustment of empirical therapy.202540142509
2228160.9998Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry. BACKGROUND: At present, phenotypic growth inhibition techniques are used in routine diagnostic microbiology to determine antimicrobial resistance of bacteria. Molecular techniques such as PCR are often used for confirmation but are indirect as they detect particular resistance genes. A direct technique would be able to detect the proteins of the resistance mechanism itself. In the present study targeted high resolution mass spectrometry assay was developed for the simultaneous detection of KPC, OXA-48-like, NDM, and VIM carbapenemases. METHODS: Carbapenemase specific target peptides were defined by comparing available sequences in GenBank. Selected peptide sequences were validated using 62 Klebsiella pneumoniae and Escherichia coli isolates containing: 16 KPC, 21 OXA-48-like, 16 NDM, 13 VIM genes, and 21 carbapenemase negative isolates. RESULTS: For each carbapenemase, two candidate peptides were validated. Method validation was performed in a blinded manner for all 83 isolates. All carbapenemases were detected. The majority was detected by both target peptides. All target peptides were 100% specific in the tested isolates and no peptide carry-over was detected. CONCLUSION: The applied targeted bottom-up mass spectrometry technique is able to accurately detect the four most prevalent carbapenemases in a single analysis.201931849899
2237170.9998Evaluation of Sepsis Flow Chip for identification of Gram-negative bacilli and detection of antimicrobial resistance genes directly from positive blood cultures. Blood stream infections are serious conditions associated with high morbi-mortality. In this study, the new Sepsis Flow Chip (SFC) assay for identification of Gram-negative bacteria and their antimicrobial resistance genes was evaluated in positive blood cultures (BCs). SFC is a microarray with a broad panel comprising the most frequent causative agents of sepsis and antimicrobial resistance genes associated with them. A total of 100 prospective BCs, positive for Gram-negative bacilli, were assessed in the routine of the clinical microbiology laboratory and also applying the SFC assay. Moreover, 19 BCs spiked with well-characterized enterobacterial isolates, harboring antimicrobial resistance genes, were analyzed by the latter. Among the monomicrobial BCs (90), the concordance between SFC identification and the reference method was 94.4%; however, it achieved 100% when SFC was combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry after 4-h incubation. Regarding polymicrobial BCs (10), 15 out of the 22 bacteria present (68.2%) were correctly identified, including all contained in 50% of the cultures. With regard to antimicrobial resistance genes, 98.8%, 98.9%, and 99% concordance was obtained for bla(CTX-M), bla(OXA-48), and bla(VIM), respectively, in comparison with polymerase chain reaction amplification. SFC assay gives results in only 4 h and showed a high concordance rate with the reference method. Although further evaluation studies are necessary, SFC assay implementation, together with antimicrobial stewardship programs, could contribute to improve the therapeutic approaches and to reduce the morbi-mortality, length of hospital stay, and healthcare-associated costs in patients with sepsis.201829551362
2315180.9998The Profile of Bacterial Infections in a Burn Unit during and after the COVID-19 Pandemic Period. Infections represent a major complication for burn-injured patients. The aim of this study was to highlight the changes in the incidence and antimicrobial resistance of bacterial strains isolated from burn patients, at the end of the COVID-19 pandemic, in relation to the antibiotics used during the pandemic. A comparative analysis of the demographic data and the microorganisms identified in the clinical samples of two groups of burn patients admitted to a university hospital in Romania was carried out. The first group consisted of 48 patients and the second of 69 patients, hospitalized in January-August 2020 and 2023, respectively. The bacterial species with the highest incidence were S. aureus, A. baumannii, Pseudomonas spp. The significant changes between 2023 and 2020 are reflected in the increase in the frequency of non-fermentative Gram-negative bacteria, especially S. maltophilia, and the increase in antimicrobial resistance of Pseudomonas and Klebsiella spp. Klebsiella spp. did not change in frequency (7%), but there was a significant increase in the incidence of K. pneumoniae strains with pan-drug resistant behaviour to antibiotics (40%), including colistin. The phenomenon can be explained by the selection of specimens carrying multiple resistance genes, as a result of antibiotic treatment during the COVID-19 period. The post-pandemic antimicrobial resistance detected in burn patients indicates the need for permanent surveillance of the resistance trends, primarily due to the limited therapeutic options available for these patients.202439334997
2209190.9997Concordance Between Antibiotic Resistance Genes and Susceptibility in Symptomatic Urinary Tract Infections. PURPOSE: Studies have shown that multiple genes influence antibiotic susceptibility, but the relationship between genotypic and phenotypic antibiotic susceptibility is unclear. We sought to analyze the concordance between the presence of antibiotic resistance (ABR) genes and antibiotic susceptibility results in urine samples collected from patients with symptomatic urinary tract infection (UTI). PATIENTS AND METHODS: Urine samples were collected from patients presenting to 37 geographically disparate urology clinics across the United States from July 2018 to February 2019. Multiplex polymerase chain reaction was used to detect 27 ABR genes. In samples containing at least one culturable organism at a concentration of ≥ 10(4) cells per mL, pooled antibiotic susceptibility testing (P-AST), which involves simultaneous growing all detected bacteria together in the presence of antibiotic and then measure susceptibility, was performed against 14 antibiotics. The concordance rate between the ABR genes and the P-AST results was generated for the overall group. The concordance rates for each antibiotic between monomicrobial and polymicrobial infection were compared using chi-square test. RESULTS: Results from ABR gene detection and P-AST of urine samples from 1155 patients were included in the concordance analysis. Overall, there was a 60% concordance between the presence or absence of ABR genes and corresponding antimicrobial susceptibility with a range of 49-78% across antibiotic classes. Vancomycin, meropenem, and piperacillin/tazobactam showed significantly lower concordance rates in polymicrobial infections than in monomicrobial infections. CONCLUSION: Given the 40% discordance rate, the detection of ABR genes alone may not provide reliable data to make informed clinical decisions in UTI management. However, when used in conjunction with susceptibility testing, ABR gene data can offer valuable clinical information for antibiotic stewardship.202134447256